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Sample records for cells identifies activated

  1. MicroRNA expression profiling identifies activated B cell status in chronic lymphocytic leukemia cells.

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    Shuqiang Li

    Full Text Available Chronic lymphocytic leukemia (CLL is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70(+ and IgV(H unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

  2. Tetrandrine identified in a small molecule screen to activate mesenchymal stem cells for enhanced immunomodulation.

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    Yang, Zijiang; Concannon, John; Ng, Kelvin S; Seyb, Kathleen; Mortensen, Luke J; Ranganath, Sudhir; Gu, Fangqi; Levy, Oren; Tong, Zhixiang; Martyn, Keir; Zhao, Weian; Lin, Charles P; Glicksman, Marcie A; Karp, Jeffrey M

    2016-07-26

    Pre-treatment or priming of mesenchymal stem cells (MSC) prior to transplantation can significantly augment the immunosuppressive effect of MSC-based therapies. In this study, we screened a library of 1402 FDA-approved bioactive compounds to prime MSC. We identified tetrandrine as a potential hit that activates the secretion of prostaglandin E2 (PGE2), a potent immunosuppressive agent, by MSC. Tetrandrine increased MSC PGE2 secretion through the NF-κB/COX-2 signaling pathway. When co-cultured with mouse macrophages (RAW264.7), tetrandrine-primed MSC attenuated the level of TNF-α secreted by RAW264.7. Furthermore, systemic transplantation of primed MSC into a mouse ear skin inflammation model significantly reduced the level of TNF-α in the inflamed ear, compared to unprimed cells. Screening of small molecules to pre-condition cells prior to transplantation represents a promising strategy to boost the therapeutic potential of cell therapy.

  3. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

    DEFF Research Database (Denmark)

    Berry, David; Mader, Esther; Lee, Tae Kwon;

    2015-01-01

    distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating...

  4. Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

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    Oliver N F King

    Full Text Available BACKGROUND: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. PRINCIPAL FINDINGS: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4 family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II and to modulate demethylation at the H3K9 locus in a cell-based assay. CONCLUSIONS: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

  5. Random Cell Identifiers Assignment

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    Robert Bestak

    2012-01-01

    Full Text Available Despite integration of advanced functions that enable Femto Access Points (FAPs to be deployed in a plug-and-play manner, the femtocell concept still cause several opened issues to be resolved. One of them represents an assignment of Physical Cell Identifiers (PCIs to FAPs. This paper analyses a random based assignment algorithm in LTE systems operating in diverse femtocell scenarios. The performance of the algorithm is evaluated by comparing the number of confusions for various femtocell densities, PCI ranges and knowledge of vicinity. Simulation results show that better knowledge of vicinity can significantly reduce the number of confusions events.

  6. High-content screening identifies kinase inhibitors that overcome venetoclax resistance in activated CLL cells.

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    Oppermann, Sina; Ylanko, Jarkko; Shi, Yonghong; Hariharan, Santosh; Oakes, Christopher C; Brauer, Patrick M; Zúñiga-Pflücker, Juan C; Leber, Brian; Spaner, David E; Andrews, David W

    2016-08-18

    Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax. PMID:27297795

  7. Proteomic Analysis of Macrophages: A Potential Way to Identify Novel Proteins Associated with Activation of Macrophages for Tumor Cell Killing

    Institute of Scientific and Technical Information of China (English)

    Lingbing Zhang; Haoxuan Zhu; Yanni Lun; Dongmei Yan; Leyang Yu; Bairong Du; Xun Zhu

    2007-01-01

    One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact,indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor (TNF) and nitric oxide (NO) are the two classical mediators of tumor cell death.However, evidence of discrepancy is accumulating indicating these known mediators do not appear to account for the broad and potent tumoricidal activity of macrophages. To obtain a full repertoire of tumoricidal activationassociated membrane proteins, we combined one-dimensional SDS-PAGE with capillary liquid chromatographytandem mass spectrometry (LC-MS/MS). Using this technique, we identified 454 activated macrophage specifically expressed proteins with extremely high confidence, including most known activation markers of macrophages,such as NO synthase (iNOS), Ym1, cyclooxygenase, etc. Membrane bound TNF-α was also identified on activated macrophages. However, it was also detected on thioglycolate elicited macrophages, indicating this molecule may not play a key role in conjugation-dependent tumor cell killing. In contrast, although NO has not been assigned as an effector molecule of conjugation-dependent tumoricidal pathway, iNOS was identified from membrane fraction of activated macrophages, suggesting NO may be involved in conjugation-dependent tumoricidal mechanism,because iNOS association with plasma membrane is ideally suited to deliver NO directly into the contacted tumor cells. This research provides not only new insights into macrophage conjugation-dependent tumoricidal mechanisms, but also a valuable data set of macrophage activation associated membrane proteins, thus providing better understanding of the functional mechanisms of macrophages in anti-tumor and other biological processes.

  8. Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

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    Kirsch Roy

    2012-11-01

    Full Text Available Abstract Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs. The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH families: GH11 (xylanases, GH28 (polygalacturonases or pectinases, and GH45 (β-1,4-glucanases or cellulases. Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs families as well as polygalacturonase-inhibiting proteins (PGIPs were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome

  9. High ALDH activity identifies chemotherapy-resistant Ewing's sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition.

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    Ola Awad

    Full Text Available BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy.

  10. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

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    Jonathan Göke

    2011-12-01

    Full Text Available Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-localization of the transcriptional co-activator Mediator, H3K27ac and increased expression of nearby genes in embryonic stem cells. We observe that the same loci bound by Oct4, Nanog and Sox2 in ES cells frequently drive expression in early embryonic development. Comparison of mouse and human ES cells shows that less than 5% of individual binding events for OCT4, SOX2 and NANOG are shared between species. In contrast, about 15% of combinatorial binding events and even between 53% and 63% of combinatorial binding events at enhancers active in early development are conserved. Our analysis suggests that the combination of OCT4, SOX2 and NANOG binding is critical for transcription in ES cells and likely plays an important role for embryogenesis by binding at conserved early developmental enhancers. Our data suggests that the fast evolutionary rewiring of regulatory networks mainly affects individual binding events, whereas "gene regulatory hotspots" which are bound by multiple factors and active in multiple tissues throughout early development are under stronger evolutionary constraints.

  11. ABCB5 Identifies Immunoregulatory Dermal Cells

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    Tobias Schatton

    2015-09-01

    Full Text Available Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5 identifies dermal immunoregulatory cells (DIRCs capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1. Purified Abcb5+ DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5+ DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

  12. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development.

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    Jonathan Göke; Marc Jung; Sarah Behrens; Lukas Chavez; Sean O'Keeffe; Bernd Timmermann; Hans Lehrach; James Adjaye; Martin Vingron

    2011-01-01

    Transcription factors are proteins that regulate gene expression by binding to cis-regulatory sequences such as promoters and enhancers. In embryonic stem (ES) cells, binding of the transcription factors OCT4, SOX2 and NANOG is essential to maintain the capacity of the cells to differentiate into any cell type of the developing embryo. It is known that transcription factors interact to regulate gene expression. In this study we show that combinatorial binding is strongly associated with co-lo...

  13. High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

    OpenAIRE

    Ola Awad; Jason T Yustein; Preeti Shah; Naheed Gul; Varalakshmi Katuri; Alison O'Neill; Yali Kong; Brown, Milton L.; Toretsky, Jeffrey A.; Loeb, David M.

    2010-01-01

    BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We h...

  14. Cell-based screening identifies the active ingredients from Traditional Chinese Medicine formula Shixiao San as the inhibitors of atherosclerotic endothelial dysfunction.

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    Xiaofan Wang

    Full Text Available In this study, we performed a phenotypic screening in human endothelial cells exposed to oxidized low density lipoprotein (an in vitro model of atherosclerotic endothelial dysfunction to identify the effective compounds in Shixiao San. After investigating the suitability and reliability of the cell-based screening method using atorvastatin as the positive control drug, this method was applied in screening Shixiao San and its extracts. The treatment of n-butanol fraction on endothelial cells exhibited stronger healing effects against oxidized low density lipoprotein-induced insult when compared with other fractions. Cell viability, the level of nitric oxide, endothelial nitric oxide synthase and endothelin-1 were measured, respectively. The assays revealed n-butanol fraction significantly elevated the survival ratio of impaired cells in culture. In parallel, n-butanol fraction exhibited the highest inhibition of inflammation. The generation of prostaglandin-2 and adhesion molecule (soluble intercellular adhesion molecule-1 was obviously declined. Furthermore, n-butanol fraction suppressed the production of reactive oxygen species and malondialdehyde, and restored the activity of superoxide dismutase. Compounds identification of the n-butanol fraction was carried out by ultra high liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. The active ingredients including quercetin-3-O-(2G-α-l-rhamnosyl-rutinoside, quercetin-3-O-neohesperidoside, isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the ability of anti-atherosclerosis after exposing on endothelial cells. The current work illustrated the pharmacology effect of Shixiao San and clearly indicated the major active components in Shixiao San. More importantly, the proposed cell-based screening method might be particularly suitable for fast evaluating the anti-atherosclerosis efficacy of Traditional Chinese Medicines and screening out the

  15. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

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    Heltemes-Harris, L M; Larson, J D; Starr, T K; Hubbard, G K; Sarver, A L; Largaespada, D A; Farrar, M A

    2016-06-30

    Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.

  16. Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

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    Heltemes-Harris, L M; Larson, J D; Starr, T K; Hubbard, G K; Sarver, A L; Largaespada, D A; Farrar, M A

    2016-06-30

    Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL. PMID:26500062

  17. Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells.

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    Lyne Khair

    2015-08-01

    Full Text Available Activation-induced cytidine deaminase (AID is required for initiation of Ig class switch recombination (CSR and somatic hypermutation (SHM of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq. We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

  18. Flavonoids Identified from Korean Scutellaria baicalensis Georgi Inhibit Inflammatory Signaling by Suppressing Activation of NF-κB and MAPK in RAW 264.7 Cells

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    Gyeong-Eun Hong

    2013-01-01

    Full Text Available Scutellaria baicalensis Georgi has been used as traditional medicine for treating inflammatory diseases, hepatitis, tumors, and diarrhea in Asia. Hence, we investigated the anti-inflammatory effect and determined the molecular mechanism of action of flavonoids isolated from Korean S. baicalensis G. in lipopolysaccharide- (LPS- stimulated RAW 264.7 macrophages. A 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay was performed to examine cytotoxicity of the flavonoids at various concentrations of 10, 40, 70, and 100 µg/mL. No cytotoxicity was observed in RAW 264.7 cells at these concentrations. Furthermore, the flavonoids decreased production of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-alpha and inhibited phosphorylation of nuclear factor-kappa B (NF-κB and mitogen-activated protein kinases (MAPKs in LPS-induced RAW 264.7 cells. Moreover, to identify the differentially expressed proteins in RAW 264.7 cells of the control, LPS-treated, and flavonoid-treated groups, two-dimensional gel electrophoresis and mass spectrometry were conducted. The identified proteins were involved in the inflammatory response and included PRKA anchor protein and heat shock protein 70 kD. These findings suggest that the flavonoids isolated from S. baicalensis G. might have anti-inflammatory effects that regulate the expression of inflammatory mediators by inhibiting the NF-κB signaling pathway via the MAPK signaling pathway in RAW 264.7 cells.

  19. Identifying Sexual Harassment: A Classroom Activity

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    Madson, Laura; Shoda, Jennifer

    2002-01-01

    We created a classroom activity to illustrate the complexity involved in identifying sexual harassment. In the activity, students decided whether 6 fictional scenarios constituted sexual harassment. The activity stimulates animated discussion, and evaluation data indicate that it received positive feedback from students and refined students'…

  20. Characterization of Ca(2+)-activated 86Rb+ fluxes in rat C6 glioma cells: a system for identifying novel IKCa-channel toxins.

    OpenAIRE

    de-Allie, F. A.; Bolsover, S. R.; Nowicky, A. V.; Strong, P N

    1996-01-01

    1. The pharmacological characteristics of a putative Ca2+ activated K+ channel (IKCa channel) in rat glioma C6 cells were studied in the presence of the Ca2+ ionophore, ionomycin and various K+ channel blockers, 86Rb+ being used as a radioisotopic tracer for K+. 2. The resting 86Rb+ influx into C6 cells was 318 +/- 20 pmol s-1. The threshold for ionomycin activation of 86Rb+ influx was approx. 100 nM. At ionomycin concentrations above the activation threshold, the initial rate of 86Rb+ influx...

  1. Identifying crucial parameter correlations maintaining bursting activity.

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    Anca Doloc-Mihu

    2014-06-01

    Full Text Available Recent experimental and computational studies suggest that linearly correlated sets of parameters (intrinsic and synaptic properties of neurons allow central pattern-generating networks to produce and maintain their rhythmic activity regardless of changing internal and external conditions. To determine the role of correlated conductances in the robust maintenance of functional bursting activity, we used our existing database of half-center oscillator (HCO model instances of the leech heartbeat CPG. From the database, we identified functional activity groups of burster (isolated neuron and half-center oscillator model instances and realistic subgroups of each that showed burst characteristics (principally period and spike frequency similar to the animal. To find linear correlations among the conductance parameters maintaining functional leech bursting activity, we applied Principal Component Analysis (PCA to each of these four groups. PCA identified a set of three maximal conductances (leak current, [Formula: see text]Leak; a persistent K current, [Formula: see text]K2; and of a persistent Na+ current, [Formula: see text]P that correlate linearly for the two groups of burster instances but not for the HCO groups. Visualizations of HCO instances in a reduced space suggested that there might be non-linear relationships between these parameters for these instances. Experimental studies have shown that period is a key attribute influenced by modulatory inputs and temperature variations in heart interneurons. Thus, we explored the sensitivity of period to changes in maximal conductances of [Formula: see text]Leak, [Formula: see text]K2, and [Formula: see text]P, and we found that for our realistic bursters the effect of these parameters on period could not be assessed because when varied individually bursting activity was not maintained.

  2. Identify multiple myeloma stem cells: Utopia?

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    Saltarella, Ilaria; Lamanuzzi, Aurelia; Reale, Antonia; Vacca, Angelo; Ria, Roberto

    2015-01-26

    Multiple myeloma (MM) is a hematologic malignancy of monoclonal plasma cells which remains incurable despite recent advances in therapies. The presence of cancer stem cells (CSCs) has been demonstrated in many solid and hematologic tumors, so the idea of CSCs has been proposed for MM, even if MM CSCs have not been define yet. The existence of myeloma CSCs with clonotypic B and clonotypic non B cells was postulated by many groups. This review aims to focus on these distinct clonotypic subpopulations and on their ability to develop and sustain MM. The bone marrow microenvironment provides to MM CSCs self-renewal, survival and drug resistance thanks to the presence of normal and cancer stem cell niches. The niches and CSCs interact each other through adhesion molecules and the interplay between ligands and receptors activates stemness signaling (Hedgehog, Wnt and Notch pathways). MM CSCs are also supposed to be responsible for drug resistance that happens in three steps from the initial cancer cell homing microenvironment-mediated to development of microenvironment-independent drug resistance. In this review, we will underline all these aspects of MM CSCs. PMID:25621108

  3. Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

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    Lara Wohlbold

    2012-08-01

    Full Text Available The cyclin-dependent kinases (CDKs that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS Cdk2 after exposure to ionizing radiation (IR enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

  4. Identified nerve cells and insect behavior.

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    Comer, C M; Robertson, R M

    2001-03-01

    Studies of insect identified neurons over the past 25 years have provided some of the very best data on sensorimotor integration; tracing information flow from sensory to motor networks. General principles have emerged that have increased the sophistication with which we now understand both sensory processing and motor control. Two overarching themes have emerged from studies of identified sensory interneurons. First, within a species, there are profound differences in neuronal organization associated with both the sex and the social experience of the individual. Second, single neurons exhibit some surprisingly rich examples of computational sophistication in terms of (a) temporal dynamics (coding superimposed upon circadian and shorter-term rhythms), and also (b) what Kenneth Roeder called "neural parsimony": that optimal information can be encoded, and complex acts of sensorimotor coordination can be mediated, by small ensembles of cells. Insect motor systems have proven to be relatively complex, and so studies of their organization typically have not yielded completely defined circuits as are known from some other invertebrates. However, several important findings have emerged. Analysis of neuronal oscillators for rhythmic behavior have delineated a profound influence of sensory feedback on interneuronal circuits: they are not only modulated by feedback, but may be substantially reconfigured. Additionally, insect motor circuits provide potent examples of neuronal restructuring during an organism's lifetime, as well as insights on how circuits have been modified across evolutionary time. Several areas where future advances seem likely to occur include: molecular genetic analyses, neuroecological syntheses, and neuroinformatics--the use of digital resources to organize databases with information on identified nerve cells and behavior. PMID:11163685

  5. Integrative analysis of the transcriptome and targetome identifies the regulatory network of miR-16: an inhibitory role against the activation of hepatic stellate cells.

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    Pan, Qin; Guo, Canjie; Sun, Chao; Fan, Jiangao; Fang, Chunhua

    2014-01-01

    Hepatic stellate cell (HSC) activation is the critical event of liver fibrosis. Abnormality of miR-16 expression induces their activation. However, the action model of miR-16 remains to be elucidated because of its multiple-targeted manner. Here, we report that miR-16 restoration exerted a wide-range impact on transcriptome (2,082 differentially expressed transcripts) of activated HSCs. Integrative analysis of both targetome (1,195 targets) and transcriptome uncovered the miR-16 regulatory network based upon bio-molecular interaction databases (BIND, BioGrid, Tranfac, and KEGG), cross database searching with iterative algorithm, Dijkstra's algorithm with greedy method, etc. Eight targets in the targetome (Map2k1, Bmpr1b, Nf1, Pik3r3, Ppp2r1a, Prkca, Smad2, and Sos2) served as key regulatory network nodes that mediate miR-16 action. A set of TFs (Sp1, Jun, Crebl, Arnt, Fos, and Nf1) was recognized to be the functional layer of key nodes, which mapped the signal of miR-16 to transcriptome. In result, the comprehensive action of miR-16 abrogated transcriptomic characteristics that determined the phenotypes of activated HSCs, including active proliferation, ECM deposition, and apoptosis resistance. Therefore, a multi-layer regulatory network based upon the integration of targetome and transcriptome may underlie the global action of miR-16, which suggesting it plays an inhibitory role in HSC activation. PMID:25227104

  6. Cell-based Assays to Identify Inhibitors of Viral Disease

    Science.gov (United States)

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong

    2009-01-01

    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  7. Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro.

    Science.gov (United States)

    Leung, Ada W Y; Hung, Stacy S; Backstrom, Ian; Ricaurte, Daniel; Kwok, Brian; Poon, Steven; McKinney, Steven; Segovia, Romulo; Rawji, Jenna; Qadir, Mohammed A; Aparicio, Samuel; Stirling, Peter C; Steidl, Christian; Bally, Marcel B

    2016-01-01

    Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both

  8. Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro.

    Directory of Open Access Journals (Sweden)

    Ada W Y Leung

    Full Text Available Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC. While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL of cisplatin (e.g. the IC10. These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were

  9. Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults.

    Science.gov (United States)

    Salaverria, Itziar; Philipp, Claudia; Oschlies, Ilske; Kohler, Christian W; Kreuz, Markus; Szczepanowski, Monika; Burkhardt, Birgit; Trautmann, Heiko; Gesk, Stefan; Andrusiewicz, Miroslaw; Berger, Hilmar; Fey, Miriam; Harder, Lana; Hasenclever, Dirk; Hummel, Michael; Loeffler, Markus; Mahn, Friederike; Martin-Guerrero, Idoia; Pellissery, Shoji; Pott, Christiane; Pfreundschuh, Michael; Reiter, Alfred; Richter, Julia; Rosolowski, Maciej; Schwaenen, Carsten; Stein, Harald; Trümper, Lorenz; Wessendorf, Swen; Spang, Rainer; Küppers, Ralf; Klapper, Wolfram; Siebert, Reiner

    2011-07-01

    The prognosis of germinal center-derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma. PMID:21487109

  10. Identifying physical activity gender differences among youth

    Science.gov (United States)

    Physical activity (PA) is an important part of a healthy lifestyle and reduces risk of certain chronic diseases. Many youth do not currently meet PA guidelines; evidence suggests that girls are less active than boys are at all ages. PA differences need to be understood, so that gender-specific inter...

  11. Methods To Identify Aptamers against Cell Surface Biomarkers

    Directory of Open Access Journals (Sweden)

    Frédéric Ducongé

    2011-09-01

    Full Text Available Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment. During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

  12. Identifying Diverse Means for Assessing Physical Activity

    Science.gov (United States)

    Perlman, Dana J.; Pearson, Phil

    2012-01-01

    Physical inactivity is of concern for the majority of age groups within the United States. Limited engagement in physical activity (PA) has been linked with an increased risk for a host of health problems, including but not limited to heart disease, diabetes and cancer. Benefits of PA are widely documented and accepted yet many people, especially…

  13. A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

    OpenAIRE

    1996-01-01

    A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and ...

  14. Gene expression profiles identify inflammatory signatures in dendritic cells.

    Directory of Open Access Journals (Sweden)

    Anna Torri

    Full Text Available Dendritic cells (DCs constitute a heterogeneous group of antigen-presenting leukocytes important in activation of both innate and adaptive immunity. We studied the gene expression patterns of DCs incubated with reagents inducing their activation or inhibition. Total RNA was isolated from DCs and gene expression profiling was performed with oligonucleotide microarrays. Using a supervised learning algorithm based on Random Forest, we generated a molecular signature of inflammation from a training set of 77 samples. We then validated this molecular signature in a testing set of 38 samples. Supervised analysis identified a set of 44 genes that distinguished very accurately between inflammatory and non inflammatory samples. The diagnostic performance of the signature genes was assessed against an independent set of samples, by qRT-PCR. Our findings suggest that the gene expression signature of DCs can provide a molecular classification for use in the selection of anti-inflammatory or adjuvant molecules with specific effects on DC activity.

  15. Comparative Proteomic Analysis of Wild-Type and SAP Domain Mutant Foot-and-Mouth Disease Virus-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication.

    Science.gov (United States)

    Zhu, Zixiang; Yang, Fan; Zhang, Keshan; Cao, Weijun; Jin, Ye; Wang, Guoqing; Mao, Ruoqing; Li, Dan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

    2015-10-01

    Leader protein (L(pro)) of foot-and-mouth disease virus (FMDV) manipulates the activities of several host proteins to promote viral replication and pathogenicity. L(pro) has a conserved protein domain SAP that is suggested to subvert interferon (IFN) production to block antiviral responses. However, apart from blocking IFN production, the roles of the SAP domain during FMDV infection in host cells remain unknown. Therefore, we identified host proteins associated with the SAP domain of L(pro) by a high-throughput quantitative proteomic approach [isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with liquid chromatography/electrospray ionization tandem mass spectrometry]. Comparison of the differentially regulated proteins in rA/FMDVΔmSAP- versus rA/FMDV-infected SK6 cells revealed 45 down-regulated and 32 up-regulated proteins that were mostly associated with metabolic, ribosome, spliceosome, and ubiquitin-proteasome pathways. The results also imply that the SAP domain has a function similar to SAF-A/B besides its potential protein inhibitor of activated signal transducer and activator of transcription (PIAS) function. One of the identified proteins UBE1 was further analyzed and displayed a novel role for the SAP domain of L(pro). Overexpression of UBE1 enhanced the replication of FMDV, and knockdown of UBE1 decreased FMDV replication. This shows that FMDV manipulates UBE1 for increased viral replication, and the SAP domain was involved in this process.

  16. A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

    International Nuclear Information System (INIS)

    A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

  17. Methods To Identify Aptamers against Cell Surface Biomarkers

    OpenAIRE

    Frédéric Ducongé; Daniel Miotto Dupont; Agnes Cibiel

    2011-01-01

    Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometr...

  18. Comparison of the Gene Expression Profiles of Human Fetal Cortical Astrocytes with Pluripotent Stem Cell Derived Neural Stem Cells Identifies Human Astrocyte Markers and Signaling Pathways and Transcription Factors Active in Human Astrocytes

    OpenAIRE

    Nasir Malik; Xiantao Wang; Sonia Shah; Efthymiou, Anastasia G.; Bin Yan; Sabrina Heman-Ackah; Ming Zhan; Mahendra Rao

    2014-01-01

    Astrocytes are the most abundant cell type in the central nervous system (CNS) and have a multitude of functions that include maintenance of CNS homeostasis, trophic support of neurons, detoxification, and immune surveillance. It has only recently been appreciated that astrocyte dysfunction is a primary cause of many neurological disorders. Despite their importance in disease very little is known about global gene expression for human astrocytes. We have performed a microarray expression anal...

  19. Identifying genes that mediate anthracyline toxicity in immune cells

    Directory of Open Access Journals (Sweden)

    Amber eFrick

    2015-04-01

    Full Text Available The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS, we identified four genome-wide significant quantitative trait loci (QTL that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01x10-8. Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05.In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Thus, further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  20. A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability

    Science.gov (United States)

    Cantisani, Maria Carmela; Parascandolo, Alessia; Perälä, Merja; Allocca, Chiara; Fey, Vidal; Sahlberg, Niko; Merolla, Francesco; Basolo, Fulvio; Laukkanen, Mikko O.; Kallioniemi, Olli Pekka; Santoro, Massimo; Castellone, Maria Domenica

    2016-01-01

    RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets. PMID:27058903

  1. Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell.

    Science.gov (United States)

    Seale, Patrick; Ishibashi, Jeff; Holterman, Chet; Rudnicki, Michael A

    2004-11-15

    Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD-/- myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD-/- myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage. PMID:15501219

  2. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6.

    Science.gov (United States)

    Wang, Yanhua; Wang, Guangxiang; Cai, Jian Ping

    2016-08-01

    The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents. PMID:27658594

  3. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  4. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  5. Immobilization of cells via activated cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Markt, M.; Kas, J.; Valentova, O.; Demnerova, K.; Vodrazka, Z.

    1986-10-01

    Cell walls of Saccharomyces cerevisiae and S. uvarum were activated by periodate oxidation of vicinal diol groups in cell wall polysaccharides. The aldehyde groups thus generated allow the yeast cells to be covalently bound to modified bead cellulose or macroporous glycidyl methacrylate supports, or to enzymes such as glucose oxidase and catalase. 6 references.

  6. Computational Approaches for Mining GRO-Seq Data to Identify and Characterize Active Enhancers.

    Science.gov (United States)

    Nagari, Anusha; Murakami, Shino; Malladi, Venkat S; Kraus, W Lee

    2017-01-01

    Transcriptional enhancers are DNA regulatory elements that are bound by transcription factors and act to positively regulate the expression of nearby or distally located target genes. Enhancers have many features that have been discovered using genomic analyses. Recent studies have shown that active enhancers recruit RNA polymerase II (Pol II) and are transcribed, producing enhancer RNAs (eRNAs). GRO-seq, a method for identifying the location and orientation of all actively transcribing RNA polymerases across the genome, is a powerful approach for monitoring nascent enhancer transcription. Furthermore, the unique pattern of enhancer transcription can be used to identify enhancers in the absence of any information about the underlying transcription factors. Here, we describe the computational approaches required to identify and analyze active enhancers using GRO-seq data, including data pre-processing, alignment, and transcript calling. In addition, we describe protocols and computational pipelines for mining GRO-seq data to identify active enhancers, as well as known transcription factor binding sites that are transcribed. Furthermore, we discuss approaches for integrating GRO-seq-based enhancer data with other genomic data, including target gene expression and function. Finally, we describe molecular biology assays that can be used to confirm and explore further the function of enhancers that have been identified using genomic assays. Together, these approaches should allow the user to identify and explore the features and biological functions of new cell type-specific enhancers. PMID:27662874

  7. Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Blix Egil S

    2012-10-01

    Full Text Available Abstract Background Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL and marginal zone lymphoma (MZL patients. Methods Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR, CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling. Results Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB, p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells. Conclusions BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation

  8. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

    International Nuclear Information System (INIS)

    The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens

  9. Satellited 4q identified in amniotic fluid cells

    Energy Technology Data Exchange (ETDEWEB)

    Miller, I.; Hsieh, C.L.; Songster, G. [Stanford Univ. Medical Center, Stanford, CA (United States)] [and others

    1995-01-16

    Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father`s relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. 8 refs., 3 figs.

  10. Transcriptional Profiling of Th2 Cells Identifies Pathogenic Features Associated with Asthma.

    Science.gov (United States)

    Seumois, Grégory; Zapardiel-Gonzalo, Jose; White, Brandie; Singh, Divya; Schulten, Veronique; Dillon, Myles; Hinz, Denize; Broide, David H; Sette, Alessandro; Peters, Bjoern; Vijayanand, Pandurangan

    2016-07-15

    Allergic asthma and rhinitis are two common chronic allergic diseases that affect the lungs and nose, respectively. Both diseases share clinical and pathological features characteristic of excessive allergen-induced type 2 inflammation, orchestrated by memory CD4(+) T cells that produce type 2 cytokines (Th2 cells). However, a large majority of subjects with allergic rhinitis do not develop asthma, suggesting divergence in disease mechanisms. Because Th2 cells play a pathogenic role in both these diseases and are also present in healthy nonallergic subjects, we performed global transcriptional profiling to determine whether there are qualitative differences in Th2 cells from subjects with allergic asthma, rhinitis, and healthy controls. Th2 cells from asthmatic subjects expressed higher levels of several genes that promote their survival as well as alter their metabolic pathways to favor persistence at sites of allergic inflammation. In addition, genes that enhanced Th2 polarization and Th2 cytokine production were also upregulated in asthma. Several genes that oppose T cell activation were downregulated in asthma, suggesting enhanced activation potential of Th2 cells from asthmatic subjects. Many novel genes with poorly defined functions were also differentially expressed in asthma. Thus, our transcriptomic analysis of circulating Th2 cells has identified several molecules that are likely to confer pathogenic features to Th2 cells that are either unique or common to both asthma and rhinitis. PMID:27271570

  11. Identifying the functional contribution of the defatty-acylase activity of SIRT6.

    Science.gov (United States)

    Zhang, Xiaoyu; Khan, Saba; Jiang, Hong; Antonyak, Marc A; Chen, Xiao; Spiegelman, Nicole A; Shrimp, Jonathan H; Cerione, Richard A; Lin, Hening

    2016-08-01

    Mammalian sirtuin 6 (SIRT6) exhibits many pivotal functions and multiple enzymatic activities, but the contribution of each activity to the various functions is unclear. We identified a SIRT6 mutant (G60A) that possesses efficient defatty-acylase activity but has substantially decreased deacetylase activity in vitro and no detectable deacetylase activity in cells. The G60A mutant has a decreased ability to bind NAD(+), but the presence of fatty-acyl lysine peptides restores NAD(+) binding, explaining the retention of the defatty-acylase activity. Using this mutant, we found that the defatty-acylase activity of SIRT6 regulates the secretion of numerous proteins. Notably, many ribosomal proteins were secreted via exosomes from Sirt6 knockout mouse embryonic fibroblasts, and these exosomes increased NIH 3T3 cell proliferation compared with control exosomes. Our data indicate that distinct activities of SIRT6 regulate different pathways and that the G60A mutant is a useful tool to study the contribution of defatty-acylase activity to SIRT6's various functions. PMID:27322069

  12. Identifying tumor cell growth inhibitors by combinatorial chemistry and zebrafish assays.

    Directory of Open Access Journals (Sweden)

    Jing Xiang

    Full Text Available Cyclin-dependent kinases (CDKs play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos. We report the successful identification of a novel lead compound that displays selective inhibitory effects on CDK2 activity, cancer cell proliferation, and tumor progression in vivo. Our approaches should have general applications in developing cell proliferation inhibitors using an efficient combinatorial chemical genetic method and integrated biological assays. The novel cell growth inhibitor we identified should have potential as a cancer therapeutic agent.

  13. Divisions of Identified Parvalbumin-Expressing Basket Cells during Working Memory-Guided Decision Making.

    Science.gov (United States)

    Lagler, Michael; Ozdemir, A Tugrul; Lagoun, Sabria; Malagon-Vina, Hugo; Borhegyi, Zsolt; Hauer, Romana; Jelem, Anna; Klausberger, Thomas

    2016-09-21

    Parvalbumin-expressing basket cells tightly control cortical networks and fire remarkably stereotyped during network oscillations and simple behaviors. How can these cells support multifaceted situations with different behavioral options and complex temporal sequences? We recorded from identified parvalbumin-expressing basket cells in prefrontal cortex of freely moving rats performing a multidimensional delayed cue-matching-to-place task, juxtacellularly filled recorded neurons for unequivocal histological identification, and determined their activity during temporally structured task episodes, associative working-memory, and stimulus-guided choice behavior. We show that parvalbumin-expressing basket cells do not fire homogenously, but individual cells were recruited or inhibited during different task episodes. Firing of individual basket cells was correlated with amount of presynaptic VIP (vasoactive intestinal polypeptide)-expressing GABAergic input. Together with subsets of pyramidal neurons, activity of basket cells differentiated for sequential actions and stimulus-guided choice behavior. Thus, interneurons of the same cell type can be recruited into different neuronal ensembles with distinct firing patterns to support multi-layered cognitive computations. PMID:27593181

  14. Identifying and understanding Indigenous ways of evaluating physical activity programs.

    Science.gov (United States)

    Roberts, Erica Blue; Butler Iii, James; Green, Kerry M; Chaudhary, Kaushal Raj

    2016-01-01

    Indigenous evaluation frameworks have not been investigated in the context of American Indian and Alaska Native (AI/AN) physical activity programs, an important area given the relationship between effective physical activity programs and quality of life among these populations. To address this gap, staff members of AI/AN physical activity programs were interviewed to explore their understanding of and experiences with evaluation. Findings suggest that Indigenous evaluation is perceived as narrative and holistic, Indigenous knowledge is used in program decision making, though it is not always acknowledged as evaluation, and there is not a universally desired way to evaluate AI/AN physical activity programs. PMID:27668593

  15. A Systematic Approach to Identify Markers of Distinctly Activated Human Macrophages

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    Bayan eSudan

    2015-05-01

    Full Text Available Polarization has been a useful concept for describing activated macrophage phenotypes and gene expression profiles. However, macrophage activation status within tumors and other settings are often inferred based on only a few markers. Complicating matters for relevance to human biology, many of the best studied macrophage activation markers have been best characterized in mice and sometimes are not similarly regulated in human macrophages. To identify novel markers of activated human macrophages, gene expression profiles for human macrophages of a single donor subjected to 33 distinct activating conditions were obtained and a set of putative activation markers were subsequently evaluated in macrophages from multiple donors using integrated fluidic circuit (IFC-based RT-PCR. Using unsupervised hierarchical clustering of the microarray screen, highly-altered transcripts (>4-fold change in expression sorted the macrophage transcription profiles into two major and 13 minor clusters. Among the 1874 highly-altered transcripts, over 100 were uniquely altered in one major or two related minor clusters. IFC PCR-derived data confirmed the microarray results and to show the kinetics of expression of potential macrophage activation markers. Transcripts encoding chemokines, cytokines, and cell surface were prominent in our analyses. The activation markers identified by this study could be used to better characterize tumor-associated macrophages from biopsies as well as other macrophage populations collected from human clinical samples.

  16. Identifying high dose activities in industrial site radiography

    International Nuclear Information System (INIS)

    Although the radiation doses received by industrial radiographers in the UK have progressively fallen over the last few years, with most now receiving less than 1 mSv/y, a few still receive, relative to the rest, much higher doses. As a percentage of all radiographers the number stays surprisingly constant from year to year. This paper describes a survey to identify the work causing these doses and suggest possible solutions. The UK Central Index of Dose Information was interrogated to identify the industrial radiography companies having staff (not necessarily the same person) with doses of greater than 5mSv/y in the last three years for which information was available. This was 15 in total. The people on the staff receiving these doses were identified and a questionnaire sent to the companies concerned requesting information about their work. A general questionnaire about the operation of the company was also included. With the agreement of the company these questionnaires were followed up by a visit to the company to interviews a number of the management and the radiographers if available. Both groups were generally very open about their problems and every discussion had a positive outcome. Several areas of work/reasons for the doses have been identified. These are: pipeline radiography, ultra sound radiographers working on nuclear reactors, complex plant work often with several teams in the area, inability to retreat from the wind out equipment due to height or access problems, site pressure to not follow the best practices and a lack of appreciation when a dose was being received or, alternatively, carelessness. Some o these problem areas are very difficult to resolve. However ways in which the Health and Safety can help influence the doses have been identified together with practical suggestions radiographers could adopt. These will be reported. (author)

  17. Machine learning models identify molecules active against the Ebola virus in vitro.

    Science.gov (United States)

    Ekins, Sean; Freundlich, Joel S; Clark, Alex M; Anantpadma, Manu; Davey, Robert A; Madrid, Peter

    2015-01-01

    The search for small molecule inhibitors of Ebola virus (EBOV) has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs) with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC 50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in vitro. PMID:26834994

  18. A Potent Activator of Melanogenesis Identified from Small Molecule Screening

    OpenAIRE

    McNaughton, Brian R.; Gareiss, Peter C.; Jacobs, Stacey E.; Fricke, Alex F.; Scott, Glynis A.; Miller, Benjamin L.

    2009-01-01

    Small molecules that increase the cellular level of melanin can be used to study melanogenesis, and have therapeutic potential for melanin-related diseases such as albinism. We describe the identification of a potent activator of melanogenesis from a targeted combinatorial library. Treating melanocytes with our most active molecule results in a 1.8-fold increase in melanin, and an increase in tyrosinase-catalyzed oxidation of L-tyrosine, a key step in melanin biosynthesis.

  19. Lineage Tracing and Cell Ablation Identify a Post-Aire-Expressing Thymic Epithelial Cell Population

    Directory of Open Access Journals (Sweden)

    Todd C. Metzger

    2013-10-01

    Full Text Available Thymic epithelial cells in the medulla (mTECs play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire, a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire+ mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire− mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.

  20. Identifying cell types from spatially referenced single-cell expression datasets.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Pettit

    2014-09-01

    Full Text Available Complex tissues, such as the brain, are composed of multiple different cell types, each of which have distinct and important roles, for example in neural function. Moreover, it has recently been appreciated that the cells that make up these sub-cell types themselves harbour significant cell-to-cell heterogeneity, in particular at the level of gene expression. The ability to study this heterogeneity has been revolutionised by advances in experimental technology, such as Wholemount in Situ Hybridizations (WiSH and single-cell RNA-sequencing. Consequently, it is now possible to study gene expression levels in thousands of cells from the same tissue type. After generating such data one of the key goals is to cluster the cells into groups that correspond to both known and putatively novel cell types. Whilst many clustering algorithms exist, they are typically unable to incorporate information about the spatial dependence between cells within the tissue under study. When such information exists it provides important insights that should be directly included in the clustering scheme. To this end we have developed a clustering method that uses a Hidden Markov Random Field (HMRF model to exploit both quantitative measures of expression and spatial information. To accurately reflect the underlying biology, we extend current HMRF approaches by allowing the degree of spatial coherency to differ between clusters. We demonstrate the utility of our method using simulated data before applying it to cluster single cell gene expression data generated by applying WiSH to study expression patterns in the brain of the marine annelid Platynereis dumereilii. Our approach allows known cell types to be identified as well as revealing new, previously unexplored cell types within the brain of this important model system.

  1. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    Science.gov (United States)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  2. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  3. NSC23925, identified in a high-throughput cell-based screen, reverses multidrug resistance.

    Directory of Open Access Journals (Sweden)

    Zhenfeng Duan

    Full Text Available BACKGROUND: Multidrug resistance (MDR is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically. METHODOLOGY/PRINCIPAL FINDINGS: To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1 but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 microM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors. CONCLUSIONS/SIGNIFICANCE: The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients.

  4. Identifying the Main Driver of Active Region Outflows

    Science.gov (United States)

    Baker, D.; van Driel-Gesztelyi, L.; Mandrini, C. H.; Démoulin, P.; Murray, M. J.

    2012-08-01

    Hinode's EUV Imaging Spectrometer (EIS) has discovered ubiquitous outflows of a few to 50 km s-1 from active regions (ARs). The characteristics of these outflows are very curious in that they are most prominent at the AR boundary and appear over monopolar magnetic areas. They are linked to strong non-thermal line broadening and are stronger in hotter EUV lines. The outflows persist for at least several days. Whereas red-shifted down flows observed in AR closed loops are well understood, to date there is no general consensus for the mechanism(s) driving blue-shifted AR-related outflows. We use Hinode EIS and X-Ray Telescope observations of AR 10942 coupled with magnetic modeling to demonstrate for the first time that the outflows originate from specific locations of the magnetic topology where field lines display strong gradients of magnetic connectivity, namely quasi-separatrix layers (QSLs), or in the limit of infinitely thin QSLs, separatrices. The strongest AR outflows were found to be in the vicinity of QSL sections located over areas of strong magnetic field. We argue that magnetic reconnection at QSLs, separating closed field lines of the AR and either large-scale externally connected or ‘open’ field lines, is a viable mechanism for driving AR outflows which are potentially sources of the slow solar wind. In fact, magnetic reconnection along QSLs (including separatricies) is the first theory to explain the most puzzling characteristics of the outflows, namely their occurrence over monopolar areas at the periphery of ARs and their longevity.

  5. Anomaly detection driven active learning for identifying suspicious tracks and events in WAMI video

    Science.gov (United States)

    Miller, David J.; Natraj, Aditya; Hockenbury, Ryler; Dunn, Katherine; Sheffler, Michael; Sullivan, Kevin

    2012-06-01

    We describe a comprehensive system for learning to identify suspicious vehicle tracks from wide-area motion (WAMI) video. First, since the road network for the scene of interest is assumed unknown, agglomerative hierarchical clustering is applied to all spatial vehicle measurements, resulting in spatial cells that largely capture individual road segments. Next, for each track, both at the cell (speed, acceleration, azimuth) and track (range, total distance, duration) levels, extreme value feature statistics are both computed and aggregated, to form summary (p-value based) anomaly statistics for each track. Here, to fairly evaluate tracks that travel across different numbers of spatial cells, for each cell-level feature type, a single (most extreme) statistic is chosen, over all cells traveled. Finally, a novel active learning paradigm, applied to a (logistic regression) track classifier, is invoked to learn to distinguish suspicious from merely anomalous tracks, starting from anomaly-ranked track prioritization, with ground-truth labeling by a human operator. This system has been applied to WAMI video data (ARGUS), with the tracks automatically extracted by a system developed in-house at Toyon Research Corporation. Our system gives promising preliminary results in highly ranking as suspicious aerial vehicles, dismounts, and traffic violators, and in learning which features are most indicative of suspicious tracks.

  6. Random mitotic activities across human embryonic stem cell colonies.

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L. (Biosciences Division)

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  7. Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets

    DEFF Research Database (Denmark)

    Adriaenssens, Alice E; Svendsen, Berit; Lam, Brian Y H;

    2016-01-01

    and delta cells. METHODS: Sst-Cre mice crossed with fluorescent reporters were used to identify delta cells, while Glu-Venus (with Venus reported under the control of the Glu [also known as Gcg] promoter) mice were used to identify alpha and beta cells. Alpha, beta and delta cells were purified using flow...... cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases. RESULTS: A database was constructed of all genes...... expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused...

  8. Analysis of micronuclei and microtubule arrangement to identify aneuploidy-inducing agents in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Degrassi, F.; Pisano, C. [Centre di Genetica Evoluzionistica, Rome (Italy); Tanzarella, C.; Antoccia, A.; Battistoni, A. [Universita La Sapienza, Rome (Italy)

    1993-12-31

    The development of in vitro test methods to detect environmental agents that might induce aneuploidy is of crucial importance in genotoxicity testing. Chromosome numerical changes may arise from damage to various cell structures and activities such as spindle components or kinetochore proteins as well as from damage to the chromosomes. Therefore, the development of effective assays to identify chromosome misdistribution in mammalian cell cultures requires the contribution of different research areas such as cytogenetics, molecular biology and cell biology. Recently, we have been working at the development of an in vitro test for aneuploidy-inducing agents combining the micronucleus assay with the immunofluorescent staining of kinetochores in micronuclei (MN). The assay has been standardized by analyzing the induction of MN containing kinetochores (CREST-positive MN) after a number of agents with different mechanism of action. Subsequently, the optimization of the assay has been carried out by introducing cytochalasin-B (cyt-B) in the test protocol in order to score MN in cells that have undergone one cell cycle. Finally, with the aim of providing an understanding of the mechanisms responsible for the production of CREST-positive MN we have analyzed the cellular structures involved in mitotic division by using specific antibodies in immunofluorescence studies.

  9. Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression.

    Directory of Open Access Journals (Sweden)

    Byungwoo Ryu

    Full Text Available BACKGROUND: Gene expression profiling has revolutionized our ability to molecularly classify primary human tumors and significantly enhanced the development of novel tumor markers and therapies; however, progress in the diagnosis and treatment of melanoma over the past 3 decades has been limited, and there is currently no approved therapy that significantly extends lifespan in patients with advanced disease. Profiling studies of melanoma to date have been inconsistent due to the heterogeneous nature of this malignancy and the limited availability of informative tissue specimens from early stages of disease. METHODOLOGY/PRINCIPLE FINDINGS: In order to gain an improved understanding of the molecular basis of melanoma progression, we have compared gene expression profiles from a series of melanoma cell lines representing discrete stages of malignant progression that recapitulate critical characteristics of the primary lesions from which they were derived. Here we describe the unsupervised hierarchical clustering of profiling data from melanoma cell lines and melanocytes. This clustering identifies two distinctive molecular subclasses of melanoma segregating aggressive metastatic tumor cell lines from less-aggressive primary tumor cell lines. Further analysis of expression signatures associated with melanoma progression using functional annotations categorized these transcripts into three classes of genes: 1 Upregulation of activators of cell cycle progression, DNA replication and repair (CDCA2, NCAPH, NCAPG, NCAPG2, PBK, NUSAP1, BIRC5, ESCO2, HELLS, MELK, GINS1, GINS4, RAD54L, TYMS, and DHFR, 2 Loss of genes associated with cellular adhesion and melanocyte differentiation (CDH3, CDH1, c-KIT, PAX3, CITED1/MSG-1, TYR, MELANA, MC1R, and OCA2, 3 Upregulation of genes associated with resistance to apoptosis (BIRC5/survivin. While these broad classes of transcripts have previously been implicated in the progression of melanoma and other malignancies, the

  10. Genome wide transcriptome analysis of dendritic cells identifies genes with altered expression in psoriasis.

    Directory of Open Access Journals (Sweden)

    Kata Filkor

    Full Text Available Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS or peptidoglycan (PGN induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs upon PGN induced tolerance. Using SAGE-Seq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (Kegg analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified

  11. Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma.

    Science.gov (United States)

    Jiang, Lu; Gu, Zhao-Hui; Yan, Zi-Xun; Zhao, Xia; Xie, Yin-Yin; Zhang, Zi-Guan; Pan, Chun-Ming; Hu, Yuan; Cai, Chang-Ping; Dong, Ying; Huang, Jin-Yan; Wang, Li; Shen, Yang; Meng, Guoyu; Zhou, Jian-Feng; Hu, Jian-Da; Wang, Jin-Fen; Liu, Yuan-Hua; Yang, Lin-Hua; Zhang, Feng; Wang, Jian-Min; Wang, Zhao; Peng, Zhi-Gang; Chen, Fang-Yuan; Sun, Zi-Min; Ding, Hao; Shi, Ju-Mei; Hou, Jian; Yan, Jin-Song; Shi, Jing-Yi; Xu, Lan; Li, Yang; Lu, Jing; Zheng, Zhong; Xue, Wen; Zhao, Wei-Li; Chen, Zhu; Chen, Sai-Juan

    2015-09-01

    Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL. PMID:26192917

  12. Apples to origins: Identifying brain tumor stem cell genes by comparing transcriptomes of normal and cancer stem cells

    OpenAIRE

    Wortham, Matthew; Yan, Hai

    2012-01-01

    The mechanisms whereby medulloblastoma stem cells coordinate tumor propagation are poorly understood. Utilizing microarray analysis, Corno and colleagues draw parallels and distinctions between medulloblastoma stem cells from the Ptch+/− mouse and normal neural stem cells, identifying Ebf3 as a cancer stem cell-specific transcript critical for tumor growth.

  13. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    Science.gov (United States)

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity.

  14. Effects of active conductance distribution over dendrites on the synaptic integration in an identified nonspiking interneuron.

    Directory of Open Access Journals (Sweden)

    Akira Takashima

    Full Text Available The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs, almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under

  15. Identifying meaningful activities among elderly people with demenitia: the developing process of an observation taxonomy

    DEFF Research Database (Denmark)

    Andresen, Mette

    2014-01-01

    Abstract title Identifying meaningful activities among elderly people with demenitia: the developing process of an observation taxonomy Abstract text Background: There is a need for tools to assist staff in identifying and planning meaningful activities for elderly nursing home residents with mod......Abstract title Identifying meaningful activities among elderly people with demenitia: the developing process of an observation taxonomy Abstract text Background: There is a need for tools to assist staff in identifying and planning meaningful activities for elderly nursing home residents...... that the first draft appears useful in identifying meaningful activities. Some adjustments are suggested, i.e. more definitions on key terms such as "activity" in the manual. Conclusion: Based on the results of the testing of the first draft a second draft will be developed and tested. The draft as well...

  16. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    DEFF Research Database (Denmark)

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra;

    2014-01-01

    Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show...... that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells...... exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key...

  17. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    Science.gov (United States)

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  18. Parallel screening of FDA-approved antineoplastic drugs for identifying sensitizers of TRAIL-induced apoptosis in cancer cells

    Directory of Open Access Journals (Sweden)

    Taylor David J

    2011-11-01

    Full Text Available Abstract Background Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL and agonistic antibodies to death receptor 4 and 5 are promising candidates for cancer therapy due to their ability to induce apoptosis selectively in a variety of human cancer cells, while demonstrating little cytotoxicity in normal cells. Although TRAIL and agonistic antibodies to DR4 and DR5 are considered safe and promising candidates in cancer therapy, many malignant cells are resistant to DR-mediated, TRAIL-induced apoptosis. In the current work, we screened a small library of fifty-five FDA and foreign-approved anti-neoplastic drugs in order to identify candidates that sensitized resistant prostate and pancreatic cancer cells to TRAIL-induced apoptosis. Methods FDA-approved drugs were screened for their ability to sensitize TRAIL resistant prostate cancer cells to TRAIL using an MTT assay for cell viability. Analysis of variance was used to identify drugs that exhibited synergy with TRAIL. Drugs demonstrating the highest synergy were selected as leads and tested in different prostate and pancreatic cancer cell lines, and one immortalized human pancreatic epithelial cell line. Sequential and simultaneous dosing modalities were investigated and the annexin V/propidium iodide assay, in concert with fluorescence microscopy, was employed to visualize cells undergoing apoptosis. Results Fourteen drugs were identified as having synergy with TRAIL, including those whose TRAIL sensitization activities were previously unknown in either prostate or pancreatic cancer cells or both. Five leads were tested in additional cancer cell lines of which, doxorubicin, mitoxantrone, and mithramycin demonstrated synergy in all lines. In particular, mitoxantrone and mithramycin demonstrated significant synergy with TRAIL and led to reduction of cancer cell viability at concentrations lower than 1 μM. At these low concentrations, mitoxantrone demonstrated selectivity toward

  19. Machine learning based methodology to identify cell shape phenotypes associated with microenvironmental cues.

    Science.gov (United States)

    Chen, Desu; Sarkar, Sumona; Candia, Julián; Florczyk, Stephen J; Bodhak, Subhadip; Driscoll, Meghan K; Simon, Carl G; Dunkers, Joy P; Losert, Wolfgang

    2016-10-01

    Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a "supercell" method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment. PMID:27449947

  20. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  1. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper;

    2016-01-01

    substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (OSR1, HDGF and ccdc82) as ATM...

  2. SILAC Proteomics of Planarians Identifies Ncoa5 as a Conserved Component of Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexander Böser

    2013-11-01

    Full Text Available Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA, which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.

  3. Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme*

    Science.gov (United States)

    Evelyn, Chris R.; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L.; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-01-01

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions. PMID:25825487

  4. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

    Institute of Scientific and Technical Information of China (English)

    Bo Song; Li-Hui Wang; Hua-Xin Wang; Ren-Shu Zheng; Lei Sun; Jian-Wu Tang; Bo Wang; Xiao-Nan Cui; Li Hou; Lu Sun; Li-Min Mao; Chun-Hui Zhou; Yue Du

    2005-01-01

    AIM: In order to obtain lymphogenous metastasisassociated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip() MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics.RESULTS: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110genes were further classified into two groups: differential biological process profile and molecular function profile.CONCLUSION: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription,chaperone activity, motor activity, protein kinase activity,receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation.Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments.ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic

  5. IDENTIFYING GENES CONTROLLING FERULATE CROSS-LINKING FORMATION IN GRASS CELL WALLS

    Energy Technology Data Exchange (ETDEWEB)

    de O Buanafina, Marcia Maria

    2013-10-16

    DESCRIPTION/ABSTRACT This proposal focuses on cell wall feruloylation and our long term goal is to identify and isolate novel genes controlling feruloylation and to characterize the phenotype of mutants in this pathway, with a spotlight on cell wall properties. Currently, the genes underlying AX feruloylation have not been identified and the isolation of such genes could be of great importance in manipulating ferulates accretion to the wall. Mutation of the feruloyl transferase gene(s) should lead to less ferulates secreted to the cell wall and reduced ferulate cross-linking. Our current research is based on the hypothesis that controlling the level of total feruloylation will have a direct impact on the level of cross-linking and in turn impact biomass utility for forage and biofuel production. Our results/accomplishments for this project so far include: 1. Mutagenised Brachypodium population. We have developed EMS mutagenized populations of model grass species Brachypodium distachyon. EMS populations have been developed from over 28,000 mutagenized seeds generating 5,184 M2 families. A total of 20,793 plants have been screened and 1,233 were originally selected. 2. Selected Brachypodium mutants: Potential mutants on their levels of cell wall ferulates and cell wall AX ? have been selected from 708 M2 families. A total of 303 back-crosses to no-mutagenized parental stock have been done, followed by selfing selected genotypes in order to confirm heritability of traits and to remove extraneous mutations generated by EMS mutagenesis. We are currently growing 12 F5 and F6 populations in order to assess CW composition. If low level of ferulates are confirmed in the candidate lines selected the mutation could be altered in different in one or several kinds of genes such as genes encoding an AX feruloyl transferase; genes encoding the arabinosyl transferase; genes encoding the synthesis of the xylan backbone; genes encoding enzymes of the monolignol pathway affecting FA

  6. A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

    Directory of Open Access Journals (Sweden)

    Melissa A Metzler

    Full Text Available The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1 activates the Mist1 promoter [corrected]. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a

  7. Differentially expressed genes identified by microarray analysis following leptin treatment of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    ZHONG Li-hua; CHENG Jun; ZHU Li-ying

    2010-01-01

    Background Liver fibrosis is the process through which numerous chronic liver diseases develop into liver cirrhosis. Leptin can activate hepatic stellate cells (HSCs) and play an important role in the formation of liver fibrosis. However, the process by which leptin activates HSCs is complicated, and research on this process is limited. The aim of this study was to explore the related changes in gene expression and the control mechanisms involved in leptin activated HSCs to understand the overall mechanism of liver fibrosis development. Methods We cultivate rat HSCs, with and without stimulation by leptin, and extracted mRNA. Differentially expressed genes were detected by microarray analysis. Results The differentially expressed genes identified included six upregulated genes and six downregulated genes. The representative upregulated genes included short chain dehydrogenase (CY5/CY3=2.265) and pulmonary surfactant protein A1 (CY5/CY3=2.036). The significant downregulated gene encoded hepatic stearoyl coenzyme A desaturase 1 (SCD-1) (CY5/CY3=0.351).Conclusion Leptin might mediate the molecular biological mechanisms of liver fibrosis.

  8. Repositioning "old" drugs for new causes: identifying new inhibitors of prostate cancer cell migration and invasion.

    Science.gov (United States)

    Shah, Esha T; Upadhyaya, Akanksha; Philp, Lisa K; Tang, Tiffany; Skalamera, Dubravka; Gunter, Jennifer; Nelson, Colleen C; Williams, Elizabeth D; Hollier, Brett G

    2016-04-01

    The majority of prostate cancer (PCa) deaths occur due to the metastatic spread of tumor cells to distant organs. Currently, there is a lack of effective therapies once tumor cells have spread outside the prostate. It is therefore imperative to rapidly develop therapeutics to inhibit the metastatic spread of tumor cells. Gain of cell motility and invasive properties is the first step of metastasis and by inhibiting motility one can potentially inhibit metastasis. Using the drug repositioning strategy, we developed a cell-based multi-parameter primary screening assay to identify drugs that inhibit the migratory and invasive properties of metastatic PC-3 PCa cells. Following the completion of the primary screening assay, 33 drugs were identified from an FDA approved drug library that either inhibited migration or were cytotoxic to the PC-3 cells. Based on the data obtained from the subsequent validation studies, mitoxantrone hydrochloride, simvastatin, fluvastatin and vandetanib were identified as strong candidates that can inhibit both the migration and invasion of PC-3 cells without significantly affecting cell viability. By employing the drug repositioning strategy instead of a de novo drug discovery and development strategy, the identified drug candidates have the potential to be rapidly translated into the clinic for the management of men with aggressive forms of PCa.

  9. Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies.

    Science.gov (United States)

    Rennert, Robert C; Januszyk, Michael; Sorkin, Michael; Rodrigues, Melanie; Maan, Zeshaan N; Duscher, Dominik; Whittam, Alexander J; Kosaraju, Revanth; Chung, Michael T; Paik, Kevin; Li, Alexander Y; Findlay, Michael; Glotzbach, Jason P; Butte, Atul J; Gurtner, Geoffrey C

    2016-01-01

    Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application. PMID:27324848

  10. Telomerase activity in germline and embryonic cells of Xenopus.

    OpenAIRE

    Mantell, L L; Greider, C W

    1994-01-01

    Telomerase is a ribonucleoprotein which synthesizes telomere repeats onto chromosome ends. Telomerase activity is involved in telomere length maintenance. We used Xenopus laevis as a model system to study the expression of telomerase activity in germline cells and during early development. We identified a non-processive telomerase activity in manually dissected nuclei of Xenopus stage VI oocytes. Telomerase activity was detected throughout oogenesis and embryogenesis. Telomerase was active in...

  11. Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying B | Division of Cancer Prevention

    Science.gov (United States)

    DESCRIPTION (provided by applicant): Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying Biomarkers for Early Detection and Risk Assessment. This application addresses Program Announcement PA-09-197: Biomarkers for Early Detection of Hematopoietic Malignancies (R01). The overall aim of this project is to identify novel biomarkers that may be used to diagnose and treat patients with Langerhans Cell Histiocytosis (LCH). LCH occurs with similar frequency as other rare malignancies including Hodgkin's lymphoma and AML. |

  12. Oscope identifies oscillatory genes in unsynchronized single cell RNA-seq experiments

    OpenAIRE

    Leng, Ning; Chu, Li-Fang; Barry, Chris; Li, Yuan; Choi, Jeea; Li, Xiaomao; Jiang, Peng; Stewart, Ron M.; Thomson, James A.; Kendziorski, Christina

    2015-01-01

    Oscillatory gene expression is fundamental to mammalian development, but technologies to monitor expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applications to a number of data sets demonstrate the utility of the approach and also identify a potential artifact in the Fluidigm C1 platform.

  13. Oscope identifies oscillatory genes in unsynchronized single-cell RNA-seq experiments.

    Science.gov (United States)

    Leng, Ning; Chu, Li-Fang; Barry, Chris; Li, Yuan; Choi, Jeea; Li, Xiaomao; Jiang, Peng; Stewart, Ron M; Thomson, James A; Kendziorski, Christina

    2015-10-01

    Oscillatory gene expression is fundamental to development, but technologies for monitoring expression oscillations are limited. We have developed a statistical approach called Oscope to identify and characterize the transcriptional dynamics of oscillating genes in single-cell RNA-seq data from an unsynchronized cell population. Applying Oscope to a number of data sets, we demonstrated its utility and also identified a potential artifact in the Fluidigm C1 platform. PMID:26301841

  14. Newly identified biomarkers for detecting circulating tumor cells in lung adenocarcinoma.

    Science.gov (United States)

    Man, Yingchun; Cao, Jingyan; Jin, Shi; Xu, Gang; Pan, Bo; Shang, Lihua; Che, Dehai; Yu, Qin; Yu, Yan

    2014-01-01

    Circulating tumor cells (CTCs) have been implicated in cancer prognosis and follow up. Detection of CTCs was considered significant in cancer evaluation. However, due to the heterogeneity and rareness of CTCs, detecting them with a single maker is usually challenged with low specificity and sensitivity. Previous studies concerning CTCs detection in lung cancer mainly focused on non-small cell lung carcinoma. Currently, there is no report yet describing the CTC detection with multiple markers in lung adenocarcinoma. In this study, by employing quantitative real-time PCR, we identified four candidate genes (mRNA) that were significantly elevated in peripheral blood mononuclear cells and biopsy tissue samples from patients with lung adenocarcinoma: cytokeratin 7 (CK7), Ca(2+)-activated chloride channel-2 (CLCA2), hyaluronan-mediated motility receptor (HMMR), and human telomerase catalytic subunit (hTERT). Then, the four markers were used for CTC detection; namely, positive detection was defined if at least one of the four markers was elevated. The positive CTC detection rate was 74.0% in patients with lung adenocarcinoma while 2.2% for healthy controls, 6.3% for benign lung disease, and 48.0% for non-adenocarcinoma non-small cell lung carcinoma. Furthermore, in a three-year follow-up study, patients with an increase in the detection markers of CTCs (CK7, CLCA2, HMMR or hTERT) on day 90 after first detection had shorter survival time compared to those with a decrease. These results demonstrate that the combination of the four markers with specificity and sensitivity is of great value in lung adenocarcinoma prognosis and follow up. PMID:25175030

  15. A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    ZHAO Lu; HE Cheng-yan; LIU Yan-li; ZHOU Hong-lan; ZHOU Jin-song; SHANG De-jing; YANG Hong

    2004-01-01

    A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(Kd=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.

  16. Mechanisms of cell propulsion by active stresses

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, A E, E-mail: aec@wustl.edu [Department of Physics, Washington University, Campus Box 1105, One Brookings Drive, St. Louis, MO 63130 (United States)

    2011-07-15

    The mechanisms by which cytoskeletal flows and cell-substrate interactions interact to generate cell motion are explored by using a simplified model of the cytoskeleton as a viscous gel containing active stresses. This model yields explicit general results relating cell speed and traction forces to the distributions of active stress and cell-substrate friction. It is found that (i) the cell velocity is given by a function that quantifies the asymmetry of the active-stress distribution, (ii) gradients in cell-substrate friction can induce motion even when the active stresses are symmetrically distributed, (iii) the traction-force dipole is enhanced by protrusive stresses near the cell edges or contractile stresses near the center of the cell and (iv) the cell velocity depends biphasically on the cell-substrate adhesion strength if active stress is enhanced by adhesion. Specific experimental tests of the calculated dependences are proposed.

  17. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...

  18. Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yoshikazu Arai

    2015-01-01

    Full Text Available The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP, mercury (Hg, cotinine, selenium (Se, and octachlorodipropyl ether (S-421. Here, we used human induced pluripotent stem cells (hiPSCs to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421 on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood.

  19. A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration

    OpenAIRE

    McLaughlin, Laura M.; Xu, Hui; Carden, Sarah E.; Fisher, Samantha; Reyes, Monique; Heilshorn, Sarah C.; Monack, Denise M.

    2014-01-01

    Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involv...

  20. DRUG-REPOSITIONING SCREENING IDENTIFIED PIPERLONGUMINE AS A DIRECT STAT3 INHIBITOR WITH POTENT ACTIVITY AGAINST BREAST CANCER

    OpenAIRE

    Bharadwaj, Uddalak; Eckols, T. Kris; Kolosov, Mikhail; Kasembeli, Moses M.; Adam, Abel; Torres, David; Zhang, Xiaomei; Lacey E Dobrolecki; Wei, Wei; Lewis, Michael T; Dave, Bhuvanesh; Chang, Jenny C.; Landis, Melissa D.; Creighton, Chad J.; Mancini, Michael A.

    2014-01-01

    Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect, and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been employed to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescenc...

  1. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    Science.gov (United States)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  2. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow

    OpenAIRE

    Balazs, Alejandro B.; Fabian, Attila J.; Esmon, Charles T.; Mulligan, Richard C.

    2006-01-01

    The hematopoietic stem cell (HSC) is a unique cell type found in bone marrow, which has the capacity for both self-renewal and differentiation into all blood lineages. The identification of genes expressed specifically in HSCs may help identify gene products vital to the control of self-renewal and/or differentiation, as well as antigens capable of forming the basis for improved methods of stem cell isolation. In previous studies, we identified a number of genes that appeared to be differenti...

  3. A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity

    Directory of Open Access Journals (Sweden)

    Ana C. D’Alessio

    2015-11-01

    Full Text Available Hundreds of transcription factors (TFs are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.

  4. LGR5 and Nanog identify stem cell signature of pancreas beta cells which initiate pancreatic cancer.

    Science.gov (United States)

    Amsterdam, Abraham; Raanan, Calanit; Schreiber, Letizia; Polin, Nava; Givol, David

    2013-04-01

    Pancreas cancer, is the fourth leading cause of cancer death but its cell of origin is controversial. We compared the localization of stem cells in normal and cancerous pancreas using antibodies to the stem cell markers Nanog and LGR5. Here we show, for the first time, that LGR5 is expressed in normal pancreas, exclusively in the islets of Langerhans and it is co-localized, surprisingly, with Nanog and insulin in clusters of beta cells. In cancerous pancreas Nanog and LGR5 are expressed in the remaining islets and in all ductal cancer cells. We observed insulin staining among the ductal cancer cells, but not in metastases. This indicates that the islet's beta cells, expressing LGR5 and Nanog markers are the initiating cells of pancreas cancer, which migrated from the islets to form the ductal cancerous tissue, probably after mutation and de-differentiation. This discovery may facilitate treatment of this devastating cancer.

  5. Laser-guidance based cell detection for identifying malignant cancerous cells without any fluorescent markers

    OpenAIRE

    Ma, Zhen; Gao, Bruce Z.

    2011-01-01

    Laser guidance technique employs the optical forces generated from a focused Gaussian laser beam incident on a biological cell to trap and guide the cell along the laser propagation direction. The optical force, which determines the guidance speed, is dependent on the cellular characteristics of the cell being guided, such as size, shape, composition and morphology. Different cell populations or subpopulations can be detected without any fluorescent markers by measuring their guidance speeds....

  6. Exosomes released from Mycoplasma infected tumor cells activate inhibitory B cells.

    Directory of Open Access Journals (Sweden)

    Chenjie Yang

    Full Text Available Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity.

  7. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

    Directory of Open Access Journals (Sweden)

    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  8. Ago2 immunoprecipitation identifies predicted microRNAs in human embryonic stem cells and neural precursors.

    Directory of Open Access Journals (Sweden)

    Loyal A Goff

    Full Text Available BACKGROUND: MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: SOLiD ultra-deep sequencing identified >10(7 unique small RNAs from human embryonic stem cells (hESC and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. CONCLUSIONS/SIGNIFICANCE: Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

  9. Differentiation of immortal cells inhibits telomerase activity.

    OpenAIRE

    Sharma, H W; Sokoloski, J A; Perez, J.R.; Maltese, J Y; Sartorelli, A C; Stein, C A; Nichols, G; Khaled, Z.; Telang, N T; Narayanan, R.

    1995-01-01

    Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, t...

  10. A genomics approach to identify susceptibilities of breast cancer cells to “fever-range” hyperthermia

    International Nuclear Information System (INIS)

    Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to “fever range” hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility. To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed. Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells. These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock

  11. TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

    Science.gov (United States)

    Kirsch, Ilan R; Watanabe, Rei; O'Malley, John T; Williamson, David W; Scott, Laura-Louise; Elco, Christopher P; Teague, Jessica E; Gehad, Ahmed; Lowry, Elizabeth L; LeBoeuf, Nicole R; Krueger, James G; Robins, Harlan S; Kupper, Thomas S; Clark, Rachael A

    2015-10-01

    Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin. PMID:26446955

  12. Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

    Science.gov (United States)

    Salehi, Hamideh; Middendorp, Elodie; Panayotov, Ivan; Dutilleul, Pierre-Yves Collard; Vegh, Attila-Gergely; Ramakrishnan, Sathish; Gergely, Csilla; Cuisinier, Frederic

    2013-05-01

    Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.

  13. A Class of High-affinity Bicyclooctane G551D-CFTR Activators Identified by High Throughput Screening

    Institute of Scientific and Technical Information of China (English)

    HE Cheng-yan; ZHAO Lu; LIU Yan-li; XU Li-na; SHANG De-jing; YANG Hong

    2004-01-01

    The glycine-to-aspartic acid missense mutation at the codon 551(G551D) of the cystic fibrosis transmembrane conductance regulator(CFTR) is one of the five most frequent cystic fibrosis(CF) mutations associated with a severe CF phenotype. To explore the feasibility of pharmacological correction of disrupted activation of CFTR chloride channel caused by G551D mutation, we developed a halide-sensitive fluorescence miniassay for G551D-CFTR in Fisher rat thyroid(FRT) epithelial cells for the discovery of novel activators of G551D-CFTR. A class of bicyclooctane small molecule compounds that efficiently stimulate G551D-CFTR chloride channel activity was identified by high throughput screening via the FRT cell-based assay. This class of compounds selectively activates G551D-CFTR with a high affinity, whereas little effect of the compounds on wildtype CFTR can be seen. The discovery of a class of bicyclooctane G551D-CFTR activators will permit the analysis of structure-activity relationship of the compounds to identify ideal leads for in vivo therapeutic studies.

  14. Activation of Natural Killer cells during microbial infections

    Directory of Open Access Journals (Sweden)

    Amir eHorowitz

    2012-01-01

    Full Text Available Natural killer (NK cells are large granular lymphocytes that express a diverse array of germline encoded inhibitory and activating receptors for MHC Class I and Class I-like molecules, classical co-stimulatory ligands and cytokines. The ability of NK cells to be very rapidly activated by inflammatory cytokines, to secrete effector cytokines and to kill infected or stressed host cells, suggests that they may be among the very early responders during infection. Recent studies have also identified a small number of pathogen-derived ligands that can bind to NK cell surface receptors and directly induce their activation. Here we review recent studies that have begun to elucidate the various pathways by which viral, bacterial and parasite pathogens activate NK cells. We also consider two emerging themes of NK cell-pathogen interactions, namely their contribution to adaptive immune responses and their potential to take on regulatory and immunomodulatory functions.

  15. Telomerase activity in plasma cell dyscrasias

    OpenAIRE

    Xu, D; Zheng, C.; Bergenbrant, S; Holm, G; Björkholm, M.; Yi, Q; Gruber, A

    2001-01-01

    Activation of telomerase is essential for in vitro cellular immortalization and tumorigenesis. In the present study, we investigated telomerase activation and its implications in plasma cell dyscrasias including monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and plasma cell leukaemia (PCL). All 5 patients with MGUS exhibited normal levels of telomerase activity in their plasma cells. Elevated telomerase activity was found in the samples from 21/27 patients wi...

  16. Sleep-active cells in the cerebral cortex and their role in slow-wave activity

    OpenAIRE

    Gerashchenko, Dmitry; Wisor, Jonathan P.; Kilduff, Thomas S.

    2011-01-01

    We recently identified neurons in the cerebral cortex that become activated during sleep episodes with high slow-wave activity (SWA). The distinctive properties of these neurons are the ability to produce nitric oxide and their long-range projections within the cortex. In this review, we discuss how these characteristics of sleep-active cells could be relevant to SWA production in the cortex. We also discuss possible models of the role of nNOS cells in SWA production.

  17. Tim-3: An activation marker and activation limiter of innate immune cells

    Directory of Open Access Journals (Sweden)

    Gencheng eHan

    2013-12-01

    Full Text Available Tim-3 was initially identified on activated Th1, Th17, and Tc1 cells and induces T cell death or exhaustion after binding to its ligand, Gal-9. The observed relationship between dysregulated Tim-3 expression on T cells and the progression of many clinical diseases has identified this molecule as an important target for intervention in adaptive immunity. Recent data have shown that it also plays critical roles in regulating the activities of macrophages, monocytes, dendritic cells, mast cells, natural killer cells, and endothelial cells. Although the underlying mechanisms remain unclear, dysregulation of Tim-3 expression on these innate immune cells leads to an excessive or inhibited inflammatory response and subsequent autoimmune damage or viral or tumor evasion. In this review, we focus on the expression and function of Tim-3 on innate immune cells and discuss 1 how Tim-3 is expressed and regulated on different innate immune cells; 2 how it affects the activity of different innate immune cells; and 3 how dysregulated Tim-3 expression on innate immune cells affects adaptive immunity and disease progression. Tim-3 is involved in the optimal activation of innate immune cells through its varied expression. A better understanding of the physiopathological role of the Tim-3 pathway in innate immunity will shed new light on the pathogenesis of clinical diseases, such as autoimmune diseases, chronic viral infections, and cancer, and suggest new approaches to intervention.

  18. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  19. Tim-3 identifies exhausted follicular helper T cells in breast cancer patients.

    Science.gov (United States)

    Zhu, Shiguang; Lin, Jun; Qiao, Guangdong; Wang, Xingmiao; Xu, Yanping

    2016-09-01

    Breast cancer is the most common cancer diagnosed in women worldwide. Although a series of treatment options have improved the overall 5-year survival rate to 90%, individual responses still vary from patient to patient. New evidence suggested that the infiltration of CXCL13-expressing CD4(+) follicular helper cells (Tfh) in breast tumor predicted better survival. Here, we examined the regulation of Tfh function in breast cancer patients in depth. We found that the frequencies of circulating Tfh cells were not altered in breast cancer patients compared to healthy controls. However, the expression of PD-1 and Tim-3 in Tfh cells was significantly elevated in breast cancer patients. Interestingly, we observed a preferential upregulation of PD-1 in Tim-3(+) Tfh cells compared to Tim-3(-) Tfh cells. Coexpression of PD-1 and Tim-3 is typically a hallmark of functional exhaustion in chronic virus infections and tumor. To examine whether Tim-3(+) identifies exhausted Tfh cells, we stimulated Tfh cells with anti-CD3/CD28, and found that Tim-3(+) T cells expressed reduced frequencies of chemokine CXCL13 and cytokine interleukin 21 (IL-21), and contained fewer proliferating cells, than Tim-3(-) Tfh cells. Compared to those cocultured with Tim-3(-) Tfh cells, naive B cells cocultured with Tim-3(+) Tfh cells resulted in significantly less IgM, IgG and IgA production after 12 day incubation, demonstrating a reduction in Tim-3(+) Tfh-mediated B cell help. Moreover, the frequencies of Tim-3(+) Tfh cells in resected breast tumor were further upregulated than autologous blood, suggesting a participation of Tim-3(+) Tfh cells in tumor physiology. Overall, the data presented here provided new insight in the regulation of Tfh cells in breast cancer patients. PMID:27156907

  20. Gametocytocidal screen identifies novel chemical classes with Plasmodium falciparum transmission blocking activity.

    Directory of Open Access Journals (Sweden)

    Natalie G Sanders

    Full Text Available Discovery of transmission blocking compounds is an important intervention strategy necessary to eliminate and eradicate malaria. To date only a small number of drugs that inhibit gametocyte development and thereby transmission from the mosquito to the human host exist. This limitation is largely due to a lack of screening assays easily adaptable to high throughput because of multiple incubation steps or the requirement for high gametocytemia. Here we report the discovery of new compounds with gametocytocidal activity using a simple and robust SYBR Green I- based DNA assay. Our assay utilizes the exflagellation step in male gametocytes and a background suppressor, which masks the staining of dead cells to achieve healthy signal to noise ratio by increasing signal of viable parasites and subtracting signal from dead parasites. By determining the contribution of exflagellation to fluorescent signal and using appropriate cutoff values, we were able to screen for gametocytocidal compounds. After assay validation and optimization, we screened an FDA approved drug library of approximately 1500 compounds, as well as the 400 compound MMV malaria box and identified 44 gametocytocidal compounds with sub to low micromolar IC50s. Major classes of compounds with gametocytocidal activity included quaternary ammonium compounds with structural similarity to choline, acridine-like compounds similar to quinacrine and pyronaridine, as well as antidepressant, antineoplastic, and anthelminthic compounds. Top drug candidates showed near complete transmission blocking in membrane feeding assays. This assay is simple, reproducible and demonstrated robust Z-factor values at low gametocytemia levels, making it amenable to HTS for identification of novel and potent gametocytocidal compounds.

  1. Cell division activity during apical hook development

    NARCIS (Netherlands)

    Raz, V.; Koornneef, M.

    2001-01-01

    Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells gro

  2. Perturbation-expression analysis identifies RUNX1 as a regulator of human mammary stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Ethan S Sokol

    2015-04-01

    Full Text Available The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS. We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.

  3. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    OpenAIRE

    Li Zou; Fahad K. Kidwai; Ross A. Kopher; Jason Motl; Cory A. Kellum; Jennifer J. Westendorf; Dan S. Kaufman

    2015-01-01

    Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow me...

  4. Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma.

    Science.gov (United States)

    Syed, Nazia; Barbhuiya, Mustafa A; Pinto, Sneha M; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T S Keshava; Kumar, M Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

  5. Integrative Functional Genomics Analysis of Sustained Polyploidy Phenotypes in Breast Cancer Cells Identifies an Oncogenic Profile for GINS2

    Directory of Open Access Journals (Sweden)

    Juha K. Rantala

    2010-11-01

    Full Text Available Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication, and possibly through polyploid intermediate states. To identify molecular processes contributing to development of aneuploidy, we used a cell spot microarray technique to identify genes inducing polyploidy and/or allowing maintenance of polyploid cell growth in breast cancer cells. Of 5760 human genes screened, 177 were found to induce severe DNA content alterations on prolonged transient silencing. Association with response to DNA damage stimulus and DNA repair was found to be the most enriched cellular processes among the candidate genes. Functional validation analysis of these genes highlighted GINS2 as the highest ranking candidate inducing polyploidy, accumulation of endogenous DNA damage, and impairing cell proliferation on inhibition. The cell growth inhibition and induction of polyploidy by suppression of GINS2 was verified in a panel of breast cancer cell lines. Bioinformatic analysis of published gene expression and DNA copy number studies of clinical breast tumors suggested GINS2 to be associated with the aggressive characteristics of a subgroup of breast cancers in vivo. In addition, nuclear GINS2 protein levels distinguished actively proliferating cancer cells suggesting potential use of GINS2 staining as a biomarker of cell proliferation as well as a potential therapeutic target.

  6. Selective activity of deguelin identifies therapeutic targets for androgen receptor-positive breast cancer.

    Science.gov (United States)

    Robles, Andrew J; Cai, Shengxin; Cichewicz, Robert H; Mooberry, Susan L

    2016-06-01

    Triple-negative breast cancers (TNBC) are aggressive malignancies with no effective targeted therapies. Recent gene expression profiling of these heterogeneous cancers and the classification of cell line models now allows for the identification of compounds with selective activities against molecular subtypes of TNBC. The natural product deguelin was found to have selective activity against MDA-MB-453 and SUM-185PE cell lines, which both model the luminal androgen receptor (LAR) subtype of TNBC. Deguelin potently inhibited proliferation of these cells with GI50 values of 30 and 61 nM, in MDA-MB-453 and SUM-185PE cells, respectively. Deguelin had exceptionally high selectivity, 197 to 566-fold, for these cell lines compared to cell lines representing other TNBC subtypes. Deguelin's mechanisms of action were investigated to determine how it produced these potent and selective effects. Our results show that deguelin has dual activities, inhibiting PI3K/Akt/mTOR signaling, and decreasing androgen receptor levels and nuclear localization. Based on these data, we hypothesized that the combination of the mTOR inhibitor rapamycin and the antiandrogen enzalutamide would have efficacy in LAR models. Rapamycin and enzalutamide showed additive effects in MDA-MB-453 cells, and both drugs had potent antitumor efficacy in a LAR xenograft model. These results suggest that the combination of antiandrogens and mTOR inhibitors might be an effective strategy for the treatment of androgen receptor-expressing TNBC. PMID:27255535

  7. Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics

    International Nuclear Information System (INIS)

    Background: Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia. Materials and methods: A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDuDD) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible. Results: All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDuDD there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was

  8. Proteomic analysis of cell lines to identify the irinotecan resistance proteins

    Indian Academy of Sciences (India)

    Xing-Chen Peng; Feng-Ming Gong; Meng Wei; X I Chen; Y E Chen; K E Cheng; Feng Gao; Feng Xu; FENG Bi; Ji-Yan Liu

    2010-12-01

    Chemotherapeutic drug resistance is a frequent cause of treatment failure in colon cancer patients. Several mechanisms have been implicated in drug resistance. However, they are not sufficient to exhaustively account for this resistance emergence. In this study, two-dimensional gel electrophoresis (2-DE) and the PDQuest software analysis were applied to compare the differential expression of irinotecan-resistance-associated protein in human colon adenocarcinoma LoVo cells and irinotecan-resistant LoVo cells (LoVo/irinotecan). The differential protein dots were excised and analysed by ESI-Q-TOF mass spectrometry (MS). Fifteen proteins were identified, including eight proteins with decreased expression and seven proteins with increased expression. The identified known proteins included those that function in diverse biological processes such as cellular transcription, cell apoptosis, electron transport/redox regulation, cell proliferation/differentiation and retinol metabolism pathways. Identification of such proteins could allow improved understanding of the mechanisms leading to the acquisition of chemoresistance.

  9. Resolving Tumor Heterogeneity: Genes Involved in Chordoma Cell Development Identified by Low-Template Analysis of Morphologically Distinct Cells

    Science.gov (United States)

    Wagner, Karin; Meditz, Katharina; Kolb, Dagmar; Feichtinger, Julia; Thallinger, Gerhard G.; Quehenberger, Franz; Liegl-Atzwanger, Bernadette; Rinner, Beate

    2014-01-01

    The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology. PMID:24503940

  10. Resolving tumor heterogeneity: genes involved in chordoma cell development identified by low-template analysis of morphologically distinct cells.

    Directory of Open Access Journals (Sweden)

    Amin El-Heliebi

    Full Text Available The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold and U-CH1 (3.7-fold cells. The mannosyltransferase ALG11 (695-fold and the phosphatase subunit PPP2CB (18.6-fold were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.

  11. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    Science.gov (United States)

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    support the use of this experimental approach to systematically identify cell pathways and molecular mechanisms involved in tick-pathogen interactions. Data are available via ProteomeXchange with identifier PXD002181.

  12. Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression.

    Science.gov (United States)

    Wissing, Michel D; Dadon, Tikva; Kim, Eunice; Piontek, Klaus B; Shim, Joong S; Kaelber, Nadine S; Liu, Jun O; Kachhap, Sushant K; Nelkin, Barry D

    2014-07-01

    Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice. PMID:24841903

  13. A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration.

    Science.gov (United States)

    McLaughlin, Laura M; Xu, Hui; Carden, Sarah E; Fisher, Samantha; Reyes, Monique; Heilshorn, Sarah C; Monack, Denise M

    2014-04-01

    Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration. PMID:24599496

  14. Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila

    Directory of Open Access Journals (Sweden)

    Xiankun Zeng

    2015-02-01

    Full Text Available The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further developed functional networks that regulate ISC self-renewal, ISC proliferation, ISC maintenance of diploid status, ISC survival, ISC-to-enterocyte (EC lineage differentiation, and ISC-to-enteroendocrine (EE lineage differentiation. By comparing regulators among ISCs, female germline stem cells, and neural stem cells, we found that factors related to basic stem cell cellular processes are commonly required in all stem cells, and stem-cell-specific, niche-related signals are required only in the unique stem cell type. Our findings provide valuable insights into stem cell maintenance and lineage-specific differentiation.

  15. Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila

    OpenAIRE

    Xiankun Zeng; Lili Han; Shree Ram Singh; Hanhan Liu; Ralph A. Neumüller; Dong Yan; Yanhui Hu; Ying Liu; Wei Liu; Xinhua Lin; Steven X. Hou

    2015-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. By integrating these genes into publicly available interaction databases, we further deve...

  16. Genome-wide RNAi Screen Identifies Networks Involved in Intestinal Stem Cell Regulation in Drosophila

    OpenAIRE

    Zeng, Xiankun; Han, Lili; Singh, Shree Ram; Liu, Hanhan; Neumüller, Ralph A.; Yan, Dong; Hu, Yanhui; Liu, Ying; Liu, Wei; Lin, Xinhua; Steven X Hou

    2015-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult animals and maintained by intestinal stem cells (ISCs) in both Drosophila and mammals. To comprehensively identify genes and pathways that regulate ISC fates, we performed a genome-wide transgenic RNAi screen in adult Drosophila intestine and identified 405 genes that regulate ISC maintenance and lineage-specific differentiation. Through integrating these genes into publicly available interaction databases, we further...

  17. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

    Science.gov (United States)

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-01-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  18. A molecular screening approach to identify and characterize inhibitors of glioblastoma stem cells

    OpenAIRE

    Visnyei, Koppany; Onodera, Hideyuki; Damoiseaux, Robert; Saigusa, Kuniyasu; Petrosyan, Syuzanna; De Vries, David; Ferrari, Denise; Saxe, Jonathan; Panosyan, Eduard H.; Masterman-Smith, Michael; Mottahedeh, Jack; Bradley, Kenneth A.; Huang, Jing; Sabatti, Chiara; Nakano, Ichiro

    2011-01-01

    Glioblastoma multiforme (GBM) is amongst the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells amongst which a tumor initiating and treatment-resistant subpopulation, here termed GBM stem cells (GSC), have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GSC. The paradigm uses a tissue cult...

  19. Machine learning models identify molecules active against the Ebola virus in vitro [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2016-01-01

    Full Text Available The search for small molecule inhibitors of Ebola virus (EBOV has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in

  20. Machine learning models identify molecules active against the Ebola virus in vitro [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2015-10-01

    Full Text Available The search for small molecule inhibitors of Ebola virus (EBOV has led to several high throughput screens over the past 3 years. These have identified a range of FDA-approved active pharmaceutical ingredients (APIs with anti-EBOV activity in vitro and several of which are also active in a mouse infection model. There are millions of additional commercially-available molecules that could be screened for potential activities as anti-EBOV compounds. One way to prioritize compounds for testing is to generate computational models based on the high throughput screening data and then virtually screen compound libraries. In the current study, we have generated Bayesian machine learning models with viral pseudotype entry assay and the EBOV replication assay data. We have validated the models internally and externally. We have also used these models to computationally score the MicroSource library of drugs to select those likely to be potential inhibitors. Three of the highest scoring molecules that were not in the model training sets, quinacrine, pyronaridine and tilorone, were tested in vitro and had EC50 values of 350, 420 and 230 nM, respectively. Pyronaridine is a component of a combination therapy for malaria that was recently approved by the European Medicines Agency, which may make it more readily accessible for clinical testing. Like other known antimalarial drugs active against EBOV, it shares the 4-aminoquinoline scaffold. Tilorone, is an investigational antiviral agent that has shown a broad array of biological activities including cell growth inhibition in cancer cells, antifibrotic properties, α7 nicotinic receptor agonist activity, radioprotective activity and activation of hypoxia inducible factor-1. Quinacrine is an antimalarial but also has use as an anthelmintic. Our results suggest data sets with less than 1,000 molecules can produce validated machine learning models that can in turn be utilized to identify novel EBOV inhibitors in

  1. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors

    Directory of Open Access Journals (Sweden)

    Zsuzsanna eSasvari

    2014-08-01

    Full Text Available To combat viral infections, plants possess innate and adaptive immune pathways, such as RNA silencing, R gene and recessive gene-mediated resistance mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF are also involved in limiting plant virus replication. This review discusses novel CIRFs with antiviral functions, many of them RNA-binding proteins or affecting the RNA binding activities of viral replication proteins. The CIRFs against tombusviruses have been identified in yeast (Saccharomyces cerevisiae, which is developed as an advanced model organism. Grouping of the identified CIRFs based on their known cellular functions and subcellular localization in yeast reveals that TBSV replication is limited by a wide variety of host gene functions. Yeast proteins with the highest connectivity in the network map include the well-characterized Xrn1p 5’-3’ exoribonuclease, Act1p actin protein and Cse4p centromere protein. The protein network map also reveals an important interplay between the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. These restriction factors frequently target the RNA-binding region in the viral replication proteins, thus interfering with the recruitment of the viral RNA for replication and the assembly of the membrane-bound viral replicase. Although many of the characterized CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function as guardians of the cellular Hsp70 chaperone system, which is subverted efficiently by TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones.

  2. Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-d-ribose Oxidase.

    Science.gov (United States)

    Batt, Sarah M; Cacho Izquierdo, Monica; Castro Pichel, Julia; Stubbs, Christopher J; Vela-Glez Del Peral, Laura; Pérez-Herrán, Esther; Dhar, Neeraj; Mouzon, Bernadette; Rees, Mike; Hutchinson, Jonathan P; Young, Robert J; McKinney, John D; Barros Aguirre, David; Ballell, Lluis; Besra, Gurdyal S; Argyrou, Argyrides

    2015-12-11

    We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase. PMID:27623058

  3. Microarray identifies ADAM family members as key responders to TGF-β1 in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Walls Dermot

    2006-09-01

    Full Text Available Abstract The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF remain elusive. Transforming Growth Factor beta 1(TGF-β1 is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549 in response to TGF-β1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-β1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-β1, suggesting a potential role for these molecules in ECM accumulation in IPF.

  4. ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas.

    Science.gov (United States)

    Adams, April; Warner, Kristy; Pearson, Alexander T; Zhang, Zhaocheng; Kim, Hong Sun; Mochizuki, Daiki; Basura, Gregory; Helman, Joseph; Mantesso, Andrea; Castilho, Rogério M; Wicha, Max S; Nör, Jacques E

    2015-09-29

    A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

  5. Active cell mechanics: Measurement and theory.

    Science.gov (United States)

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  6. Exome Genotyping Identifies Pleiotropic Variants Associated with Red Blood Cell Traits

    DEFF Research Database (Denmark)

    Chami, Nathalie; Chen, Ming-Huei; Slater, Andrew J;

    2016-01-01

    Red blood cell (RBC) traits are important heritable clinical biomarkers and modifiers of disease severity. To identify coding genetic variants associated with these traits, we conducted meta-analyses of seven RBC phenotypes in 130,273 multi-ethnic individuals from studies genotyped on an exome ar...

  7. Genome-wide association study identifies multiple susceptibility loci for diffuse large B cell lymphoma

    NARCIS (Netherlands)

    Cerhan, James R.; Berndt, Sonja I.; Vijai, Joseph; Ghesquières, Hervé; McKay, James; Wang, Sophia S.; Wang, Zhaoming; Yeager, Meredith; Conde, Lucia; De Bakker, Paul I W; Nieters, Alexandra; Cox, David; Burdett, Laurie; Monnereau, Alain; Flowers, Christopher R.; De Roos, Anneclaire J.; Brooks-Wilson, Angela R.; Lan, Qing; Severi, Gianluca; Melbye, Mads; Gu, Jian; Jackson, Rebecca D.; Kane, Eleanor; Teras, Lauren R.; Purdue, Mark P.; Vajdic, Claire M.; Spinelli, John J.; Giles, Graham G.; Albanes, Demetrius; Kelly, Rachel S.; Zucca, Mariagrazia; Bertrand, Kimberly A.; Zeleniuch-Jacquotte, Anne; Lawrence, Charles; Hutchinson, Amy; Zhi, Degui; Habermann, Thomas M.; Link, Brian K.; Novak, Anne J.; Dogan, Ahmet; Asmann, Yan W.; Liebow, Mark; Thompson, Carrie A.; Ansell, Stephen M.; Witzig, Thomas E.; Weiner, George J.; Veron, Amelie S.; Zelenika, Diana; Tilly, Hervé; Haioun, Corinne; Molina, Thierry Jo; Hjalgrim, Henrik; Glimelius, Bengt; Adami, Hans Olov; Bracci, Paige M.; Riby, Jacques; Smith, Martyn T.; Holly, Elizabeth A.; Cozen, Wendy; Hartge, Patricia; Morton, Lindsay M.; Severson, Richard K.; Tinker, Lesley F.; North, Kari E.; Becker, Nikolaus; Benavente, Yolanda; Boffetta, Paolo; Brennan, Paul; Foretova, Lenka; Maynadie, Marc; Staines, Anthony; Lightfoot, Tracy; Crouch, Simon; Smith, Alex; Roman, Eve; Diver, W. Ryan; Offit, Kenneth; Zelenetz, Andrew; Klein, Robert J.; Villano, Danylo J.; Zheng, Tongzhang; Zhang, Yawei; Holford, Theodore R.; Kricker, Anne; Turner, Jenny; Southey, Melissa C.; Clavel, Jacqueline; Virtamo, Jarmo; Weinstein, Stephanie; Riboli, Elio; Vineis, Paolo; Kaaks, Rudolph; Trichopoulos, Dimitrios; Vermeulen, Roel C H; Boeing, Heiner; Tjonneland, Anne; Angelucci, Emanuele; Di Lollo, Simonetta; Rais, Marco; Birmann, Brenda M.; Laden, Francine; Giovannucci, Edward; Kraft, Peter; Huang, Jinyan; Ma, Baoshan; Ye, Yuanqing; Chiu, Brian C H; Sampson, Joshua; Liang, Liming; Park, Ju Hyun; Chung, Charles C.; Weisenburger, Dennis D.; Chatterjee, Nilanjan; Fraumeni, Joseph F.; Slager, Susan L.; Wu, Xifeng; De Sanjose, Silvia; Smedby, Karin E.; Salles, Gilles; Skibola, Christine F.; Rothman, Nathaniel; Chanock, Stephen J.

    2014-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common lymphoma subtype and is clinically aggressive. To identify genetic susceptibility loci for DLBCL, we conducted a meta-analysis of 3 new genome-wide association studies (GWAS) and 1 previous scan, totaling 3,857 cases and 7,666 controls of Euro

  8. SABER: a computational method for identifying active sites for new reactions.

    Science.gov (United States)

    Nosrati, Geoffrey R; Houk, K N

    2012-05-01

    A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified.

  9. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    Science.gov (United States)

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.

  10. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    Science.gov (United States)

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells. PMID:27175912

  11. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  12. Identifying predictors of activity based anorexia susceptibility in diverse genetic rodent populations.

    Directory of Open Access Journals (Sweden)

    Eneda Pjetri

    Full Text Available Animal studies are very useful in detection of early disease indicators and in unravelling the pathophysiological processes underlying core psychiatric disorder phenotypes. Early indicators are critical for preventive and efficient treatment of progressive psychiatric disorders like anorexia nervosa. Comparable to physical hyperactivity observed in anorexia nervosa patients, in the activity-based anorexia rodent model, mice and rats express paradoxical high voluntary wheel running activity levels when food restricted. Eleven inbred mouse strains and outbred Wistar WU rats were exposed to the activity-based anorexia model in search of identifying susceptibility predictors. Body weight, food intake and wheel running activity levels of each individual mouse and rat were measured. Mouse strains and rats with high wheel running activity levels during food restriction exhibited accelerated body weight loss. Linear mixed models for repeated measures analysis showed that baseline wheel running activity levels preceding the scheduled food restriction phase strongly predicted activity-based anorexia susceptibility (mice: Beta  =  -0.0158 (±0.003 SE, P<0.0001; rats: Beta  =  -0.0242 (±0.004 SE, P<0.0001 compared to other baseline parameters. These results suggest that physical activity levels play an important role in activity-based anorexia susceptibility in different rodent species with genetically diverse background. These findings support previous retrospective studies on physical activity levels in anorexia nervosa patients and indicate that pre-morbid physical activity levels could reflect an early indicator for disease severity.

  13. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

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    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  14. High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations

    Science.gov (United States)

    Manshian, Bella B.; Munck, Sebastian; Agostinis, Patrizia; Himmelreich, Uwe; Soenen, Stefaan J.

    2015-09-01

    A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

  15. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  16. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

    NARCIS (Netherlands)

    Merlos-Suarez, A.; Barriga, F.M.; Jung, P.; Iglesias, M.; Cespedes, M.V.; Rossell, D.; Sevillano, M.; Hernando-Momblona, X.; da Silva-Diz, V.; Munoz, P.; Clevers, H.; Sancho, E.; Mangues, R.; Batlle, E.

    2011-01-01

    A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradu

  17. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon bacterial stimulation. CD3-CD56+ NK cells were isolated from...... having engulfed bacteria, stimulated the growth of the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various lactobacilli have the capacity to activate NK cells in vitro, in a monocyte dependent...

  18. Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.

    Directory of Open Access Journals (Sweden)

    Kai Connie Wu

    Full Text Available Nuclear factor erythroid 2-related factor 2 (Nrf2 is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96% increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones. In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.

  19. An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition

    Directory of Open Access Journals (Sweden)

    Gardiner Kristin L

    2011-10-01

    Full Text Available Abstract Background The evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents. Methods One fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D under varying conditions and the outcome of competition measured over 14 days by flow cytometry. Results We demonstrate that ascorbic acid (vitamin C can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus. Conclusions This model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.

  20. CD271 as a marker to identify mesenchymal stem cells fromdiverse sources before culture

    Institute of Scientific and Technical Information of China (English)

    María álvarez-Viejo; Yolanda Menéndez-Menéndez; Jesús Otero-Hernández

    2015-01-01

    Mesenchymal stem cells, due to their characteristicsare ideal candidates for cellular therapy. Currently,in culture these cells are defined by their adherenceto plastic, specific surface antigen expression andmultipotent differentiation potential. However, thein vivo identification of mesenchymal stem cells,before culture, is not so well established. Pre-cultureidentification markers would ensure higher purity thanthat obtained with selection based on adherence toplastic. Up until now, CD271 has been described asthe most specific marker for the characterization andpurification of human bone marrow mesenchymal stemcells. This marker has been shown to be specificallyexpressed by these cells. Thus, CD271 has beenproposed as a versatile marker to selectively isolatedand expand multipotent mesenchymal stem cells withboth immunosuppressive and lymphohematopoieticengraftment-promoting properties. This reviewfocuses on this marker, specifically on identificationof mesenchymal stem cells from different tissues.Literature revision suggests that CD271 should not bedefined as a universal marker to identify mesenchymalstem cells before culture from different sources. In thecase of bone marrow or adipose tissue, CD271 couldbe considered a quite suitable marker; however thismarker seems to be inadequate for the isolation ofmesenchymal stem cells from other tissues such asumbilical cord blood or wharton's jelly among others.

  1. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    Science.gov (United States)

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  2. Drug-repositioning screening identified piperlongumine as a direct STAT3 inhibitor with potent activity against breast cancer.

    Science.gov (United States)

    Bharadwaj, U; Eckols, T K; Kolosov, M; Kasembeli, M M; Adam, A; Torres, D; Zhang, X; Dobrolecki, L E; Wei, W; Lewis, M T; Dave, B; Chang, J C; Landis, M D; Creighton, C J; Mancini, M A; Tweardy, D J

    2015-03-12

    Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been used to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. PL inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that PL directly inhibited binding of Stat3 to its phosphotyrosyl peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as Janus kinases, Src kinase family members or receptor tyrosine kinases. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient-derived xenografts. PL's antitumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 has a role. PMID:24681959

  3. An Efficient Method to Identify Conditionally Activated Transcription Factors and their Corresponding Signal Transduction Pathway Segments

    Directory of Open Access Journals (Sweden)

    Haiyan Hu

    2009-11-01

    Full Text Available A signal transduction pathway (STP is a cascade composed of a series of signal transferring steps, which often activate one or more transcription factors (TFs to control the transcription of target genes. Understanding signaling pathways is important to our understanding of the molecular mechanisms of disease. Many condition-annotated pathways have been deposited in public databases. However, condition-annotated pathways are far from complete, considering the large number of possible conditions. Computational methods to assist in the identification of conditionally activated pathways are greatly needed. In this paper, we propose an efficient method to identify conditionally activated pathway segments starting from the identification of conditionally activated TFs, by incorporating protein-DNA binding data, gene expression data and protein interaction data. Applying our methods on several microarray datasets, we have discovered many significantly activated TFs and their corresponding pathway segments, which are supported by evidence in the literature.

  4. A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions

    Directory of Open Access Journals (Sweden)

    Robert Kraft

    2013-01-01

    The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the ‘filagree’ phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity. The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the ‘beads-on-a-string’ defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

  5. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

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    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  6. Measurement of myeloid cell immune suppressive activity.

    Science.gov (United States)

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  7. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    Science.gov (United States)

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-01

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

  8. Genome-wide DNA methylation analysis identifies hypomethylated genes regulated by FOXP3 in human regulatory T cells.

    Science.gov (United States)

    Zhang, Yuxia; Maksimovic, Jovana; Naselli, Gaetano; Qian, Junyan; Chopin, Michael; Blewitt, Marnie E; Oshlack, Alicia; Harrison, Leonard C

    2013-10-17

    Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

  9. Dissemination in athymic nude mice of lacZ transfected small cell lung cancer cells identified by X-gal staining

    DEFF Research Database (Denmark)

    Rømer, M U; Christiansen, J; Brünner, N;

    1995-01-01

    with the chromogenic substrate X-gal. lacZ expressing cells were investigated after subcutaneous (s.c.) inoculation and intravenous (i.v.) injection. The X-gal detection of beta-D-galactosidase activity proved to be a rapid and easy means for specific and highly sensitive identification of metastases. All primary s.......c. tumors stained by X-gal. The primary tumors of GLC-2 regularly demonstrated local invasive growth and produced multiple metastases in several organs. In contrast, primary DMS 456 tumors only occasionally demonstrated local invasion and very rarely generated secondary foci. No experimental metastases were......The small cell lung cancer cell lines GLC-2 and DMS 456 were genetically labeled with the lacZ gene and examined for invasive and metastatic potential in META/Bom nude mice. The lacZ gene encodes the enzyme beta-D- galactosidase, and cells expressing this enzyme were identified by staining...

  10. Activated protein C modulates the proinflammatory activity of dendritic cells

    Directory of Open Access Journals (Sweden)

    Matsumoto T

    2015-05-01

    Full Text Available Takahiro Matsumoto,1,2* Yuki Matsushima,1* Masaaki Toda,1 Ziaurahman Roeen,1 Corina N D'Alessandro-Gabazza,1,5 Josephine A Hinneh,1 Etsuko Harada,1,3 Taro Yasuma,4 Yutaka Yano,4 Masahito Urawa,1,5 Tetsu Kobayashi,5 Osamu Taguchi,5 Esteban C Gabazza1 1Department of Immunology, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, 2BONAC Corporation, BIO Factory 4F, Fukuoka, 3Iwade Research Institute of Mycology, 4Department of Endocrinology, Diabetes and Metabolism, 5Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu, Mie Prefecture, Japan *These authors contributed equally to this work Background: Previous studies have demonstrated the beneficial activity of activated protein C in allergic diseases including bronchial asthma and rhinitis. However, the exact mechanism of action of activated protein C in allergies is unclear. In this study, we hypothesized that pharmacological doses of activated protein C can modulate allergic inflammation by inhibiting dendritic cells. Materials and methods: Dendritic cells were prepared using murine bone marrow progenitor cells and human peripheral monocytes. Bronchial asthma was induced in mice that received intratracheal instillation of ovalbumin-pulsed dendritic cells. Results: Activated protein C significantly increased the differentiation of tolerogenic plasmacytoid dendritic cells and the secretion of type I interferons, but it significantly reduced lipopolysaccharide-mediated maturation and the secretion of inflammatory cytokines in myeloid dendritic cells. Activated protein C also inhibited maturation and the secretion of inflammatory cytokines in monocyte-derived dendritic cells. Activated protein C-treated dendritic cells were less effective when differentiating naïve CD4 T-cells from Th1 or Th2 cells, and the cellular effect of activated protein C was mediated by its receptors. Mice that received adoptive transfer of activated protein C

  11. Single-Cell Profiling of Epigenetic Modifiers Identifies PRDM14 as an Inducer of Cell Fate in the Mammalian Embryo

    Directory of Open Access Journals (Sweden)

    Adam Burton

    2013-11-01

    Full Text Available Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

  12. A novel pattern mining approach for identifying cognitive activity in EEG based functional brain networks.

    Science.gov (United States)

    Thilaga, M; Vijayalakshmi, R; Nadarajan, R; Nandagopal, D

    2016-06-01

    The complex nature of neuronal interactions of the human brain has posed many challenges to the research community. To explore the underlying mechanisms of neuronal activity of cohesive brain regions during different cognitive activities, many innovative mathematical and computational models are required. This paper presents a novel Common Functional Pattern Mining approach to demonstrate the similar patterns of interactions due to common behavior of certain brain regions. The electrode sites of EEG-based functional brain network are modeled as a set of transactions and node-based complex network measures as itemsets. These itemsets are transformed into a graph data structure called Functional Pattern Graph. By mining this Functional Pattern Graph, the common functional patterns due to specific brain functioning can be identified. The empirical analyses show the efficiency of the proposed approach in identifying the extent to which the electrode sites (transactions) are similar during various cognitive load states.

  13. Active Gel Model of Amoeboid Cell Motility

    CERN Document Server

    Callan-Jones, A C

    2013-01-01

    We develop a model of amoeboid cell motility based on active gel theory. Modeling the motile apparatus of a eukaryotic cell as a confined layer of finite length of poroelastic active gel permeated by a solvent, we first show that, due to active stress and gel turnover, an initially static and homogeneous layer can undergo a contractile-type instability to a polarized moving state in which the rear is enriched in gel polymer. This agrees qualitatively with motile cells containing an actomyosin-rich uropod at their rear. We find that the gel layer settles into a steadily moving, inhomogeneous state at long times, sustained by a balance between contractility and filament turnover. In addition, our model predicts an optimal value of the gel-susbstrate adhesion leading to maximum layer speed, in agreement with cell motility assays. The model may be relevant to motility of cells translocating in complex, confining environments that can be mimicked experimentally by cell migration through microchannels.

  14. High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

    Science.gov (United States)

    Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

    2014-06-01

    The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

  15. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    OpenAIRE

    Achkar, Jacqueline M.; Laetitia Cortes; Pascal Croteau; Corey Yanofsky; Marija Mentinova; Isabelle Rajotte; Michael Schirm; Yiyong Zhou; Ana Paula Junqueira-Kipnis; Kasprowicz, Victoria O.; Michelle Larsen; René Allard; Joanna Hunter; Eustache Paramithiotis

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry...

  16. Individual human brain areas can be identified from their characteristic spectral activation fingerprints

    OpenAIRE

    Keitel, Anne; Gross, Joachim

    2016-01-01

    The human brain can be parcellated into diverse anatomical areas. We investigated whether rhythmic brain activity in these areas is characteristic and can be used for automatic classification. To this end, resting-state MEG data of 22 healthy adults was analysed. Power spectra of 1-s long data segments for atlas-defined brain areas were clustered into spectral profiles (“fingerprints”), using k-means and Gaussian mixture (GM) modelling. We demonstrate that individual areas can be identified f...

  17. Microarray Analysis in a Cell Death Resistant Glioma Cell Line to Identify Signaling Pathways and Novel Genes Controlling Resistance and Malignancy

    Directory of Open Access Journals (Sweden)

    Janina Seznec

    2011-06-01

    Full Text Available Glioblastoma multiforme (GBM is a lethal type of cancer mainly resistant to radio- and chemotherapy. Since the tumor suppressor p53 functions as a transcription factor regulating the expression of genes involved in growth inhibition, DNA repair and apoptosis, we previously assessed whether specific differences in the modulation of gene expression are responsible for the anti-tumor properties of a dominant positive p53, chimeric tumor suppressor (CTS-1. CTS-1 is based on the sequence of p53 and designed to resist various mechanisms of inactivation which limit the activity of p53. To identify CTS-1-regulated cell death-inducing genes, we generated a CTS-1-resistant glioma cell line (229R. We used Affymetrix whole-genome microarray expression analysis to analyze alterations in gene expression and identified a variety of CTS-1 regulated genes involved in cancer-linked processes. 313 genes were differentially expressed in Adeno-CTS-1 (Ad-CTS-1-infected and 700 genes in uninfected 229R cells compared to matching parental cells. Ingenuity Pathway Analysis (IPA determined a variety of differentially expressed genes in Ad-CTS-1-infected cells that were members of the intracellular networks with central tumor-involved players such as nuclear factor kappa B (NF-κB, protein kinase B (PKB/AKT or transforming growth factor beta (TGF-β. Differentially regulated genes include secreted factors as well as intracellular proteins and transcription factors regulating not only cell death, but also processes such as tumor cell motility and immunity. This work gives an overview of the pathways differentially regulated in the resistant versus parental glioma cells and might be helpful to identify candidate genes which could serve as targets to develop novel glioma specific therapy strategies.

  18. Microarray Analysis in a Cell Death Resistant Glioma Cell Line to Identify Signaling Pathways and Novel Genes Controlling Resistance and Malignancy

    Energy Technology Data Exchange (ETDEWEB)

    Seznec, Janina; Naumann, Ulrike, E-mail: ulrike.naumann@uni-tuebingen.de [Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie-Institute for Clinical Brain Research and Center Neurology, University of Tuebingen, Otfried-Mueller-Str. 27, Tuebingen 72076 (Germany)

    2011-06-27

    Glioblastoma multiforme (GBM) is a lethal type of cancer mainly resistant to radio- and chemotherapy. Since the tumor suppressor p53 functions as a transcription factor regulating the expression of genes involved in growth inhibition, DNA repair and apoptosis, we previously assessed whether specific differences in the modulation of gene expression are responsible for the anti-tumor properties of a dominant positive p53, chimeric tumor suppressor (CTS)-1. CTS-1 is based on the sequence of p53 and designed to resist various mechanisms of inactivation which limit the activity of p53. To identify CTS-1-regulated cell death-inducing genes, we generated a CTS-1-resistant glioma cell line (229R). We used Affymetrix whole-genome microarray expression analysis to analyze alterations in gene expression and identified a variety of CTS-1 regulated genes involved in cancer-linked processes. 313 genes were differentially expressed in Adeno-CTS-1 (Ad-CTS-1)-infected and 700 genes in uninfected 229R cells compared to matching parental cells. Ingenuity Pathway Analysis (IPA) determined a variety of differentially expressed genes in Ad-CTS-1-infected cells that were members of the intracellular networks with central tumor-involved players such as nuclear factor kappa B (NF-κB), protein kinase B (PKB/AKT) or transforming growth factor beta (TGF-β). Differentially regulated genes include secreted factors as well as intracellular proteins and transcription factors regulating not only cell death, but also processes such as tumor cell motility and immunity. This work gives an overview of the pathways differentially regulated in the resistant versus parental glioma cells and might be helpful to identify candidate genes which could serve as targets to develop novel glioma specific therapy strategies.

  19. Transcriptome Analysis of Individual Stromal Cell Populations Identifies Stroma-Tumor Crosstalk in Mouse Lung Cancer Model

    Directory of Open Access Journals (Sweden)

    Hyejin Choi

    2015-02-01

    Full Text Available Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments.

  20. Transcriptome analysis of individual stromal cell populations identifies stroma-tumor crosstalk in mouse lung cancer model.

    Science.gov (United States)

    Choi, Hyejin; Sheng, Jianting; Gao, Dingcheng; Li, Fuhai; Durrans, Anna; Ryu, Seongho; Lee, Sharrell B; Narula, Navneet; Rafii, Shahin; Elemento, Olivier; Altorki, Nasser K; Wong, Stephen T C; Mittal, Vivek

    2015-02-24

    Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments. PMID:25704820

  1. Cellular, Molecular Consequences of Peroxisome Proliferator- Activated Receptor-δ Activation in Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sara Vignati

    2006-10-01

    Full Text Available Peroxisome proliferator-activated receptor-δ (PPAR-δ is a ligand-activated transcription factor. In addition to its canonical role in lipid, glucose metabolism, PPAR-δ controls cell proliferation, death, differentiation in several tissues. Here we have examined the expression of PPAR-δ in ovarian tumors, the cellular, molecular consequences of its activation in ovarian cancer cells. PPAR-δ was expressed in a large number of epithelial ovarian tumors, cell lines. The PPAR-δ lig, ciglitazone inhibited the growth, clonogenic survival of ovarian cancer cells, inducing cell cycle arrest, cell death. Growth inhibition by ciglitazone was reversed by the PPAR-δ antagonist GW9662, indicating the involvement of PPAR-δ- dependent mechanisms. Microarray-based gene profiling revealed complex changes in the transcriptional program of ovarian cancer cells on treatment with ciglitazone, identified multiple pathways that may contribute to PPAR-δ ligands' antitumor activity. Genes upregulated by ciglitazone were predominantly associated with metabolic, differentiation, tumorsuppressor pathways, whereas downregulated genes were involved in cell proliferation, cell cycle, cell organization, steroid biosynthesis. Collectively, our data indicate that PPAR-δ activation by selective agonists is a valid strategy for ovarian cancer therapy, prevention, should be tested alone, in combination with other anticancer drugs.

  2. Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone

    Science.gov (United States)

    Terayama, Hayato; Hirai, Shuichi; Naito, Munekazu; Qu, Ning; Katagiri, Chiaki; Nagahori, Kenta; Hayashi, Shogo; Sasaki, Hiraku; Moriya, Shota; Hiramoto, Masaki; Miyazawa, Keisuke; Hatayama, Naoyuki; Li, Zhong-Lian; Sakabe, Kou; Matsushita, Masayuki; Itoh, Masahiro

    2016-01-01

    There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body’s immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies. PMID:27752123

  3. A phase synchronization clustering algorithm for identifying interesting groups of genes from cell cycle expression data

    Directory of Open Access Journals (Sweden)

    Tcha Hong

    2008-01-01

    Full Text Available Abstract Background The previous studies of genome-wide expression patterns show that a certain percentage of genes are cell cycle regulated. The expression data has been analyzed in a number of different ways to identify cell cycle dependent genes. In this study, we pose the hypothesis that cell cycle dependent genes are considered as oscillating systems with a rhythm, i.e. systems producing response signals with period and frequency. Therefore, we are motivated to apply the theory of multivariate phase synchronization for clustering cell cycle specific genome-wide expression data. Results We propose the strategy to find groups of genes according to the specific biological process by analyzing cell cycle specific gene expression data. To evaluate the propose method, we use the modified Kuramoto model, which is a phase governing equation that provides the long-term dynamics of globally coupled oscillators. With this equation, we simulate two groups of expression signals, and the simulated signals from each group shares their own common rhythm. Then, the simulated expression data are mixed with randomly generated expression data to be used as input data set to the algorithm. Using these simulated expression data, it is shown that the algorithm is able to identify expression signals that are involved in the same oscillating process. We also evaluate the method with yeast cell cycle expression data. It is shown that the output clusters by the proposed algorithm include genes, which are closely associated with each other by sharing significant Gene Ontology terms of biological process and/or having relatively many known biological interactions. Therefore, the evaluation analysis indicates that the method is able to identify expression signals according to the specific biological process. Our evaluation analysis also indicates that some portion of output by the proposed algorithm is not obtainable by the traditional clustering algorithm with

  4. Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV gag p24 protein.

    Directory of Open Access Journals (Sweden)

    Leonia Bozzacco

    Full Text Available Mass Spectrometry (MS is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC on antigen presenting cells (APC. We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs. This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ, indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.

  5. Bcl11b mutations identified in murine lymphomas increase the proliferation rate of hematopoietic progenitor cells

    Directory of Open Access Journals (Sweden)

    Söderkvist Peter

    2007-10-01

    Full Text Available Abstract Background The telomeric region of mouse chromosome 12 has previously shown frequent allelic loss in murine lymphoma. The Bcl11b gene has been identified and suggested as a candidate tumor suppressor gene within this region. In this study, we aimed to elucidate whether Bcl11b is mutated in lymphomas with allelic loss, and whether the mutations we detected conferred any effect on cell proliferation and apoptosis. Methods Mouse lymphomas induced by 1,3-butadiene or 2',3'-dideoxycytidine were analysed for mutations in the Bcl11b gene using single strand conformation analysis and direct DNA sequencing. Effects on cell proliferation by the detected mutations were studied by expressing wild-type and mutant Bcl11b in the cytokine-dependent hematopoietic progenitor cell line FDC-P1, lacking endogenous Bcl11b expression. Results Missense and frameshift (FS mutations were identified in 7 of 47 tumors (15%. Interestingly, all mutations were found between amino acids 778–844 which encode the three C-terminal DNA-binding zinc fingers. In FDC-P1 cells, wild-type Bcl11b suppressed cell proliferation, whereas the mutated versions (S778N, K828T, Y844C and FS823 enhanced proliferation several-fold. Conclusion The genetic alterations detected in this study suggest that the three C-terminal zinc fingers of Bcl11b are important for the DNA-binding. Cell proliferation was suppressed by overexpression of wild-type Bcl11b but enhanced by mutant Bcl11b, indicating that these mutations may be an important contributing factor to lymphomagenesis in a subset of tumors.

  6. A molecular screening approach to identify and characterize inhibitors of glioblastoma stem cells.

    Science.gov (United States)

    Visnyei, Koppany; Onodera, Hideyuki; Damoiseaux, Robert; Saigusa, Kuniyasu; Petrosyan, Syuzanna; De Vries, David; Ferrari, Denise; Saxe, Jonathan; Panosyan, Eduard H; Masterman-Smith, Michael; Mottahedeh, Jack; Bradley, Kenneth A; Huang, Jing; Sabatti, Chiara; Nakano, Ichiro; Kornblum, Harley I

    2011-10-01

    Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. PMID:21859839

  7. Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas

    OpenAIRE

    Paschke, Ralf; Jaeschke, Holger; Mueller, Sandra; Eszlinger, Markus

    2010-01-01

    Abstract Objective: Constitutively activating mutations of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas. Design: Mutations L677V and T620I identified in hot thyroid carc...

  8. Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

    Directory of Open Access Journals (Sweden)

    Jill M Brooks

    2016-04-01

    Full Text Available Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501, as well as subdominant responses through common class I alleles (e.g. B7 and C*0304. Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.

  9. Synthetic Lethality Screen Identifies RPS6KA2 as Modifier of Epidermal Growth Factor Receptor Activity in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Nada Milosevic

    2013-12-01

    Full Text Available Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK/extracellular-signal regulated kinase (ERK signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

  10. Activity driven fluctuations in living cells

    CERN Document Server

    Fodor, É; Gov, N S; Visco, P; Weitz, D A; van Wijland, F

    2015-01-01

    We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.

  11. Sox17-Mediated XEN Cell Conversion Identifies Dynamic Networks Controlling Cell-Fate Decisions in Embryo-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela C.H. McDonald

    2014-10-01

    Full Text Available Little is known about the gene regulatory networks (GRNs distinguishing extraembryonic endoderm (ExEn stem (XEN cells from those that maintain the extensively characterized embryonic stem cell (ESC. An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

  12. Quantitative proteomics identifies central players in erlotinib resistance of the non-small cell lung cancer cell line HCC827

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Lund, Rikke Raaen; Beck, Hans Christian;

    , but surprisingly not of AKT and FOXO1/3a, indicating that AKT is the main signaling hub for survival. Also Erk1/2 phsphorylation is pertained although at decreased levels. Conclusions: In conclusion, cancer-related networks such as proliferation and apoptosis were found to be regulated, supporting the validity...... by a secondary mutation (T790M) in EGFR. Importantly, a majority of resistance cases are still unexplained. Our aim is to identify novel resistance mechanisms in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20...... or other EGFR or KRAS mutations, potentiating the identification of novel resistance mechanisms. We identified 2875 cytoplasmic proteins present in all 4 cell lines. Of these 87, 56 and 23 are upregulated >1.5 fold; and 117, 72 and 32 are downregulated >1.5 fold, respectively, in the 3 resistant clones...

  13. Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors

    Science.gov (United States)

    Cho, Edward H.; Wendel, Marco; Luttgen, Madelyn; Yoshioka, Craig; Marrinucci, Dena; Lazar, Daniel; Schram, Ethan; Nieva, Jorge; Bazhenova, Lyudmila; Morgan, Alison; Ko, Andrew H.; Korn, W. Michael; Kolatkar, Anand; Bethel, Kelly; Kuhn, Peter

    2012-02-01

    Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

  14. Identifying barriers to patient acceptance of active surveillance: content analysis of online patient communications.

    Directory of Open Access Journals (Sweden)

    Mark V Mishra

    Full Text Available OBJECTIVES: Qualitative research aimed at identifying patient acceptance of active surveillance (AS has been identified as a public health research priority. The primary objective of this study was to determine if analysis of a large-sample of anonymous internet conversations (ICs could be utilized to identify unmet public needs regarding AS. METHODS: English-language ICs regarding prostate cancer (PC treatment with AS from 2002-12 were identified using a novel internet search methodology. Web spiders were developed to mine, aggregate, and analyze content from the world-wide-web for ICs centered on AS. Collection of ICs was not restricted to any specific geographic region of origin. NLP was used to evaluate content and perform a sentiment analysis. Conversations were scored as positive, negative, or neutral. A sentiment index (SI was subsequently calculated according to the following formula to compare temporal trends in public sentiment towards AS: [(# Positive IC/#Total IC-(#Negative IC/#Total IC x 100]. RESULTS: A total of 464 ICs were identified. Sentiment increased from -13 to +2 over the study period. The increase sentiment has been driven by increased patient emphasis on quality-of-life factors and endorsement of AS by national medical organizations. Unmet needs identified in these ICs include: a gap between quantitative data regarding long-term outcomes with AS vs. conventional treatments, desire for treatment information from an unbiased specialist, and absence of public role models managed with AS. CONCLUSIONS: This study demonstrates the potential utility of online patient communications to provide insight into patient preferences and decision-making. Based on our findings, we recommend that multidisciplinary clinics consider including an unbiased specialist to present treatment options and that future decision tools for AS include quantitative data regarding outcomes after AS.

  15. Identifying induced seismicity in active tectonic regions: A case study of the San Joaquin Basin, California

    Science.gov (United States)

    Aminzadeh, F.; Göbel, T.

    2013-12-01

    Understanding the connection between petroleum-industry activities, and seismic event occurrences is essential to monitor, quantify, and mitigate seismic risk. While many studies identified anthropogenically-induced seismicity in intraplate regions where background seismicity rates are generally low, little is known about how to distinguish naturally occurring from induced seismicity in active tectonic regions. Further, it is not clear how different oil and gas operational parameters impact the frequency and magnitude of the induced seismic events. Here, we examine variations in frequency-size and spatial distributions of seismicity within the Southern Joaquin basin, an area of both active petroleum production and active fault systems. We analyze a newly available, high-quality, relocated earthquake catalog (Hauksson et al. 2012). This catalog includes many seismic events with magnitudes up to M = 4.5 within the study area. We start by analyzing the overall quality and consistence of the seismic catalog, focusing on temporal variations in seismicity rates and catalog completeness which could indicate variations in network sensitivity. This catalog provides relatively homogeneous earthquake recordings after 1981, enabling us to compare seismicity rates before and after the beginning of more pervasive petroleum-industry activities, for example, hydraulic-fracturing and waste-water disposals. We conduct a limited study of waste-water disposal wells to establish a correlation between seismicity statistics (i.e. rate changes, fractal dimension, b-value) within specific regions and anthropogenic influences. We then perform a regional study, to investigate spatial variations in seismicity statistics which are then correlated to oil field locations and well densities. In order to distinguish, predominantly natural seismicity from induced seismicity, we perform a spatial mapping of b-values and fractal dimensions of earthquake hypocenters. Seismic events in the proximity to

  16. Identifying active methane-oxidizers in thawed Arctic permafrost by proteomics

    Science.gov (United States)

    Lau, C. M.; Stackhouse, B. T.; Chourey, K.; Hettich, R. L.; Vishnivetskaya, T. A.; Pfiffner, S. M.; Layton, A. C.; Mykytczuk, N. C.; Whyte, L.; Onstott, T. C.

    2012-12-01

    The rate of CH4 release from thawing permafrost in the Arctic has been regarded as one of the determining factors on future global climate. It is uncertain how indigenous microorganisms would interact with such changing environmental conditions and hence their impact on the fate of carbon compounds that are sequestered in the cryosol. Multitudinous studies of pristine surface cryosol (top 5 cm) and microcosm experiments have provided growing evidence of effective methanotrophy. Cryosol samples corresponding to active layer were sampled from a sparsely vegetated, ice-wedge polygon at the McGill Arctic Research Station at Axel Heiberg Island, Nunavut, Canada (N79°24, W90°45) before the onset of annual thaw. Pyrosequencing of 16S rRNA gene indicated the occurrence of methanotroph-containing bacterial families as minor components (~5%) in pristine cryosol including Bradyrhizobiaceae, Methylobacteriaceae and Methylocystaceae within alpha-Proteobacteria, and Methylacidiphilaceae within Verrucomicrobia. The potential of methanotrophy is supported by preliminary analysis of metagenome data, which indicated putative methane monooxygenase gene sequences relating to Bradyrhizobium sp. and Pseudonocardia sp. are present. Proteome profiling in general yielded minute traces of proteins, which likely hints at dormant nature of the soil microbial consortia. The lack of specific protein database for permafrost posted additional challenge to protein identification. Only 35 proteins could be identified in the pristine cryosol and of which 60% belonged to Shewanella sp. Most of the identified proteins are known to be involved in energy metabolism or post-translational modification of proteins. Microcosms amended with sodium acetate exhibited a net methane consumption of ~65 ngC-CH4 per gram (fresh weight) of soil over 16 days of aerobic incubation at room temperature. The pH in microcosm materials remained acidic (decreased from initial 4.7 to 4.5). Protein extraction and

  17. Measuring enzyme activity in single cells

    OpenAIRE

    Kovarik, Michelle L.; Allbritton, Nancy L.

    2011-01-01

    Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range o...

  18. Syndecans: synergistic activators of cell adhesion

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1998-01-01

    Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual...... molecules modulating integrin-based adhesion....

  19. Leishmania chagasi T-cell antigens identified through a double library screen.

    Science.gov (United States)

    Martins, Daniella R A; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2006-12-01

    Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines. PMID:17000724

  20. Integrative genomic analyses identify BRF2 as a novel lineage-specific oncogene in lung squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    William W Lockwood

    2010-07-01

    Full Text Available BACKGROUND: Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes--adenocarcinoma (AC and squamous cell carcinoma (SqCC--respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome. METHODS AND FINDINGS: We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330, normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage. CONCLUSIONS: This is the first study, to our knowledge, to show that the focal amplification of a gene in

  1. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression.

    Science.gov (United States)

    Reniere, Michelle L; Whiteley, Aaron T; Portnoy, Daniel A

    2016-07-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  2. Identifying parameters in active magnetic bearing system using LFT formulation and Youla factorization

    DEFF Research Database (Denmark)

    Lauridsen, Jonas; Sekunda, André Krabdrup; Santos, Ilmar;

    2015-01-01

    In this paper, a method for identifying uncertain parameters in a rotordynamic system composed of a flexible rotating shaft, rigid discs and two radial active magnetic bearings is presented. Shaft and disc dynamics are mathematically described using a Finite Element (FE) model while magnetic...... techniques are required., The main focus of the paper relies on how to effectively identify uncertain parameters, such as stiffness and damping force coefficients of bearings and seals in rotordynamic systems. Dynamic condensation method, i.e. pseudo-modal reduction, is used to obtain a reduced order model...... of the system matrix A of the full FE model while it is represented as several elements spread over multiple rows and columns of the system matrix of the reduced model. The parametric uncertainty, for both the full and reduced FE model, is represented using Linear Fractional Transformation (LFT). In this way...

  3. Application of laser microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells

    Directory of Open Access Journals (Sweden)

    Andrea eBerruti

    2013-05-01

    Full Text Available Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intracellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells through a laser microdissection (LMD approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the arbusculated cells. Unexpectedly, one taxon was found in the arbusculated cells, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community.

  4. Lipolytic activity in adipocyte cell fractions.

    Science.gov (United States)

    Oschry, Y; Shapiro, B

    1980-05-28

    Adipocytes release only negligible amounts of free fatty acids unless stimulated, but reveal considerable lipolytic activity when homogenized. Epinephrine treatment of the cells caused only a 20-40% increase in the activity of infranatants of homogenates while raising the activity associated with the fat layer up to 10-fold. Full activity (i.e. that of intact-activated cells) could be revealed by epinephrine treatment of the homogenate as well as by sonication of the fat layer in buffer. The combination of both treatments did not yield higher activities. The fat cake contains the bulk of the potential activities which are only realized when dispersed in the aqueous phase by sonication, or upon hormone activation of the whole homogenate. Increase in activity could also be obtained by removal of most of the lipid from the fat layer by extraction with petroleum ether. Re-introduction of extracted lipid inhibited lipolysis. The active enzyme could be separated by flotation at 1.12 specific gravity. The data suggest that the lack of activity in the intact non-stimulated cell may be due to the lack of availability of the aqueous phase to the enzyme. PMID:7378439

  5. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

    Directory of Open Access Journals (Sweden)

    Leena Pohjala

    Full Text Available Chikungunya virus (CHIKV, an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2, obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs. The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV, their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate

  6. Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology.

    Science.gov (United States)

    Moreno, Marta; Fernández, Virginia; Monllau, Josep M; Borrell, Víctor; Lerin, Carles; de la Iglesia, Núria

    2015-08-11

    Neural stem cells (NSCs) reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs) that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA) of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT) and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state.

  7. Active oxygen and cell death in cereal aleurone cells.

    Science.gov (United States)

    Fath, Angelika; Bethke, Paul; Beligni, Veronica; Jones, Russell

    2002-05-01

    The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

  8. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins.

    Science.gov (United States)

    Chan, Yuk-Kit; Zhang, Huoming; Liu, Pei; Tsao, Sai-Wah; Lung, Maria Li; Mak, Nai-Ki; Ngok-Shun Wong, Ricky; Ying-Kit Yue, Patrick

    2015-10-15

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future.

  9. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit

    2015-04-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  10. Tools to identify the men with prostate cancer most appropriate for active surveillance?

    Directory of Open Access Journals (Sweden)

    Robert H Getzenberg

    2014-02-01

    Full Text Available A great deal of effort is underway in order to identify those men with prostate cancer felicitous for active surveillance with greater precision than that afforded to us today. In the manuscript by Irshad et al. the authors evaluate a novel set of genes associated with senescence and aging as tools that can provide guidance regarding the indolent nature of an individual's prostate cancer with validation using both mRNA and protein analyses. While additional studies are required to understand the full impact of these findings, the innovative approach taken enhances our understanding of distinct phenotypes of prostate cancer.

  11. Differential Aspartate Usage Identifies a Subset of Cancer Cells Particularly Dependent on OGDH

    Directory of Open Access Journals (Sweden)

    Eric L. Allen

    2016-10-01

    Full Text Available Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

  12. Genome-wide association study identifies 14 novel risk alleles associated with basal cell carcinoma.

    Science.gov (United States)

    Chahal, Harvind S; Wu, Wenting; Ransohoff, Katherine J; Yang, Lingyao; Hedlin, Haley; Desai, Manisha; Lin, Yuan; Dai, Hong-Ji; Qureshi, Abrar A; Li, Wen-Qing; Kraft, Peter; Hinds, David A; Tang, Jean Y; Han, Jiali; Sarin, Kavita Y

    2016-01-01

    Basal cell carcinoma (BCC) is the most common cancer worldwide with an annual incidence of 2.8 million cases in the United States alone. Previous studies have demonstrated an association between 21 distinct genetic loci and BCC risk. Here, we report the results of a two-stage genome-wide association study of BCC, totalling 17,187 cases and 287,054 controls. We confirm 17 previously reported loci and identify 14 new susceptibility loci reaching genome-wide significance (P<5 × 10(-8), logistic regression). These newly associated SNPs lie within predicted keratinocyte regulatory elements and in expression quantitative trait loci; furthermore, we identify candidate genes and non-coding RNAs involved in telomere maintenance, immune regulation and tumour progression, providing deeper insight into the pathogenesis of BCC. PMID:27539887

  13. Effect of newly identified hTERT-interacting proteins on telomerase activity

    Institute of Scientific and Technical Information of China (English)

    Lina Zhou; Bing Chen; Xing Hua; Ping Zhou; Lian Guo; Yong Peng; Kunhua Qiu

    2013-01-01

    There is a close relationship between telomeres-telomerase and age-related disease.Human telomerase reverse transcriptase (hTERT) is both the catalytic component of human telomerase and the rate-limiting determinant of telomerase activity.Its transcriptional regulation is the primary mode of control of telomerase activity.It is critical to find the proteins interacting with hTERT for exploring the regulatory mechanisms of the hTERT expression and the telomerase activity.In this study,the yeast two-hybrid system was used to screen the potential interactive proteins of hTERT.Six proteins were obtained,among which TSTAR,LOXL3,HKR3,and Par-4 were further confirmed as the interacting proteins of hTERT by co-immunopreci-pitation.Then the sense and antisense gene eukaryotic expression vectors containing these four genes were constructed and transfected into tumor cell lines.The correlations among the expression levels of these four proteins,the expression level of hTERT,and the telomerase activity were analyzed.Results showed that the up-regulation of TSTAR expression and down-regulation of HKR3 expression led to the increase of hTERT expression and telomerase activity,while the up-and down-regulation of LOXL3 and Par-4 expressions had no obvious effect.Our results suggested that T-STAR has a positive correlation with the telomerase activity while HKR3 may be a negative regulator.This conclusion is important to further explore the influencing factors or regulation pathways of human telomerase activity,which may be of great importance for the potential clinical application.

  14. Identifying the common interaction networks of amoeboid motility and cancer cell metastasis

    Directory of Open Access Journals (Sweden)

    Ahmed H. Zeitoun

    2012-06-01

    Full Text Available The recently analyzed genome of Naegleria gruberi, a free-living amoeboflagellate of the Heterolobosea clade, revealed a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal-, motility- and signaling-genes. This protist, which diverged from other eukaryotic lineages over a billion years ago, possesses the ability for both amoeboid and flagellar motility. In a phylogenomic comparison of two free living eukaryotes with large proteomic datasets of three metastatic tumour entities (malignant melanoma, breast- and prostate-carcinoma, we find common proteins with potential importance for cell motility and cancer cell metastasis. To identify the underlying signaling modules, we constructed for each tumour type a protein-protein interaction network including these common proteins. The connectivity within this interactome revealed specific interactions and pathways which constitute prospective points of intervention for novel anti-metastatic tumour therapies.

  15. Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.

    Science.gov (United States)

    Cao, Jingli; Navis, Adam; Cox, Ben D; Dickson, Amy L; Gemberling, Matthew; Karra, Ravi; Bagnat, Michel; Poss, Kenneth D

    2016-01-15

    In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

  16. A novel single cell method to identify the genetic composition at a single nuclear body

    Science.gov (United States)

    Anchel, David; Ching, Reagan W.; Cotton, Rachel; Li, Ren; Bazett-Jones, David P.

    2016-01-01

    Gene loci make specific associations with compartments of the nucleus (e.g. the nuclear envelope, nucleolus, and transcription factories) and this association may determine or reflect a mechanism of genetic control. With current methods, it is not possible to identify sets of genes that converge to form a “gene hub” as there is a reliance on loci-specific probes, or immunoprecipitation of a particular protein from bulk cells. We introduce a method that will allow for the identification of loci contained within the vicinity of a single nuclear body in a single cell. For the first time, we demonstrate that the DNA sequences originating from a single sub-nuclear structure in a single cell targeted by two-photon irradiation can be determined, and mapped to a particular locus. Its application to single PML nuclear bodies reveals ontologically related loci that frequently associate with each other and with PML bodies in a population of cells, and a possible nuclear body targeting role for specific transcription factor binding sites. PMID:27389808

  17. Exome Genotyping Identifies Pleiotropic Variants Associated with Red Blood Cell Traits.

    Science.gov (United States)

    Chami, Nathalie; Chen, Ming-Huei; Slater, Andrew J; Eicher, John D; Evangelou, Evangelos; Tajuddin, Salman M; Love-Gregory, Latisha; Kacprowski, Tim; Schick, Ursula M; Nomura, Akihiro; Giri, Ayush; Lessard, Samuel; Brody, Jennifer A; Schurmann, Claudia; Pankratz, Nathan; Yanek, Lisa R; Manichaikul, Ani; Pazoki, Raha; Mihailov, Evelin; Hill, W David; Raffield, Laura M; Burt, Amber; Bartz, Traci M; Becker, Diane M; Becker, Lewis C; Boerwinkle, Eric; Bork-Jensen, Jette; Bottinger, Erwin P; O'Donoghue, Michelle L; Crosslin, David R; de Denus, Simon; Dubé, Marie-Pierre; Elliott, Paul; Engström, Gunnar; Evans, Michele K; Floyd, James S; Fornage, Myriam; Gao, He; Greinacher, Andreas; Gudnason, Vilmundur; Hansen, Torben; Harris, Tamara B; Hayward, Caroline; Hernesniemi, Jussi; Highland, Heather M; Hirschhorn, Joel N; Hofman, Albert; Irvin, Marguerite R; Kähönen, Mika; Lange, Ethan; Launer, Lenore J; Lehtimäki, Terho; Li, Jin; Liewald, David C M; Linneberg, Allan; Liu, Yongmei; Lu, Yingchang; Lyytikäinen, Leo-Pekka; Mägi, Reedik; Mathias, Rasika A; Melander, Olle; Metspalu, Andres; Mononen, Nina; Nalls, Mike A; Nickerson, Deborah A; Nikus, Kjell; O'Donnell, Chris J; Orho-Melander, Marju; Pedersen, Oluf; Petersmann, Astrid; Polfus, Linda; Psaty, Bruce M; Raitakari, Olli T; Raitoharju, Emma; Richard, Melissa; Rice, Kenneth M; Rivadeneira, Fernando; Rotter, Jerome I; Schmidt, Frank; Smith, Albert Vernon; Starr, John M; Taylor, Kent D; Teumer, Alexander; Thuesen, Betina H; Torstenson, Eric S; Tracy, Russell P; Tzoulaki, Ioanna; Zakai, Neil A; Vacchi-Suzzi, Caterina; van Duijn, Cornelia M; van Rooij, Frank J A; Cushman, Mary; Deary, Ian J; Velez Edwards, Digna R; Vergnaud, Anne-Claire; Wallentin, Lars; Waterworth, Dawn M; White, Harvey D; Wilson, James G; Zonderman, Alan B; Kathiresan, Sekar; Grarup, Niels; Esko, Tõnu; Loos, Ruth J F; Lange, Leslie A; Faraday, Nauder; Abumrad, Nada A; Edwards, Todd L; Ganesh, Santhi K; Auer, Paul L; Johnson, Andrew D; Reiner, Alexander P; Lettre, Guillaume

    2016-07-01

    Red blood cell (RBC) traits are important heritable clinical biomarkers and modifiers of disease severity. To identify coding genetic variants associated with these traits, we conducted meta-analyses of seven RBC phenotypes in 130,273 multi-ethnic individuals from studies genotyped on an exome array. After conditional analyses and replication in 27,480 independent individuals, we identified 16 new RBC variants. We found low-frequency missense variants in MAP1A (rs55707100, minor allele frequency [MAF] = 3.3%, p = 2 × 10(-10) for hemoglobin [HGB]) and HNF4A (rs1800961, MAF = 2.4%, p < 3 × 10(-8) for hematocrit [HCT] and HGB). In African Americans, we identified a nonsense variant in CD36 associated with higher RBC distribution width (rs3211938, MAF = 8.7%, p = 7 × 10(-11)) and showed that it is associated with lower CD36 expression and strong allelic imbalance in ex vivo differentiated human erythroblasts. We also identified a rare missense variant in ALAS2 (rs201062903, MAF = 0.2%) associated with lower mean corpuscular volume and mean corpuscular hemoglobin (p < 8 × 10(-9)). Mendelian mutations in ALAS2 are a cause of sideroblastic anemia and erythropoietic protoporphyria. Gene-based testing highlighted three rare missense variants in PKLR, a gene mutated in Mendelian non-spherocytic hemolytic anemia, associated with HGB and HCT (SKAT p < 8 × 10(-7)). These rare, low-frequency, and common RBC variants showed pleiotropy, being also associated with platelet, white blood cell, and lipid traits. Our association results and functional annotation suggest the involvement of new genes in human erythropoiesis. We also confirm that rare and low-frequency variants play a role in the architecture of complex human traits, although their phenotypic effect is generally smaller than originally anticipated.

  18. Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

    Science.gov (United States)

    Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

    2016-09-01

    Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. PMID:27235586

  19. Using time-driven activity-based costing to identify value improvement opportunities in healthcare.

    Science.gov (United States)

    Kaplan, Robert S; Witkowski, Mary; Abbott, Megan; Guzman, Alexis Barboza; Higgins, Laurence D; Meara, John G; Padden, Erin; Shah, Apurva S; Waters, Peter; Weidemeier, Marco; Wertheimer, Sam; Feeley, Thomas W

    2014-01-01

    As healthcare providers cope with pricing pressures and increased accountability for performance, they should be rededicating themselves to improving the value they deliver to their patients: better outcomes and lower costs. Time-driven activity-based costing offers the potential for clinicians to redesign their care processes toward that end. This costing approach, however, is new to healthcare and has not yet been systematically implemented and evaluated. This article describes early time-driven activity-based costing work at several leading healthcare organizations in the United States and Europe. It identifies the opportunities they found to improve value for patients and demonstrates how this costing method can serve as the foundation for new bundled payment reimbursement approaches.

  20. Using time-driven activity-based costing to identify value improvement opportunities in healthcare.

    Science.gov (United States)

    Kaplan, Robert S; Witkowski, Mary; Abbott, Megan; Guzman, Alexis Barboza; Higgins, Laurence D; Meara, John G; Padden, Erin; Shah, Apurva S; Waters, Peter; Weidemeier, Marco; Wertheimer, Sam; Feeley, Thomas W

    2014-01-01

    As healthcare providers cope with pricing pressures and increased accountability for performance, they should be rededicating themselves to improving the value they deliver to their patients: better outcomes and lower costs. Time-driven activity-based costing offers the potential for clinicians to redesign their care processes toward that end. This costing approach, however, is new to healthcare and has not yet been systematically implemented and evaluated. This article describes early time-driven activity-based costing work at several leading healthcare organizations in the United States and Europe. It identifies the opportunities they found to improve value for patients and demonstrates how this costing method can serve as the foundation for new bundled payment reimbursement approaches. PMID:25647962

  1. Evaluating digestion efficiency in full-scale anaerobic digesters by identifying active microbial populations through the lens of microbial activity

    Science.gov (United States)

    Mei, Ran; Narihiro, Takashi; Nobu, Masaru K.; Kuroda, Kyohei; Liu, Wen-Tso

    2016-09-01

    Anaerobic digestion is a common technology to biologically stabilize wasted solids produced in municipal wastewater treatment. Its efficiency is usually evaluated by calculating the reduction in volatile solids, which assumes no biomass growth associated with digestion. To determine whether this assumption is valid and further evaluate digestion efficiency, this study sampled 35 digester sludge from different reactors at multiple time points together with the feed biomass in a full-scale water reclamation plant at Chicago, Illinois. The microbial communities were characterized using Illumina sequencing technology based on 16S rRNA and 16S rRNA gene (rDNA). 74 core microbial populations were identified and represented 58.7% of the entire digester community. Among them, active populations were first identified using the ratio of 16S rRNA and 16S rDNA (rRNA/rDNA) for individual populations, but this approach failed to generate consistent results. Subsequently, a recently proposed mass balance model was applied to calculate the specific growth rate (μ), and this approach successfully identified active microbial populations in digester (positive μ) that could play important roles than those with negative μ. It was further estimated that 82% of microbial populations in the feed sludge were digested in comparison with less than 50% calculated using current equations.

  2. Evaluating digestion efficiency in full-scale anaerobic digesters by identifying active microbial populations through the lens of microbial activity

    Science.gov (United States)

    Mei, Ran; Narihiro, Takashi; Nobu, Masaru K.; Kuroda, Kyohei; Liu, Wen-Tso

    2016-01-01

    Anaerobic digestion is a common technology to biologically stabilize wasted solids produced in municipal wastewater treatment. Its efficiency is usually evaluated by calculating the reduction in volatile solids, which assumes no biomass growth associated with digestion. To determine whether this assumption is valid and further evaluate digestion efficiency, this study sampled 35 digester sludge from different reactors at multiple time points together with the feed biomass in a full-scale water reclamation plant at Chicago, Illinois. The microbial communities were characterized using Illumina sequencing technology based on 16S rRNA and 16S rRNA gene (rDNA). 74 core microbial populations were identified and represented 58.7% of the entire digester community. Among them, active populations were first identified using the ratio of 16S rRNA and 16S rDNA (rRNA/rDNA) for individual populations, but this approach failed to generate consistent results. Subsequently, a recently proposed mass balance model was applied to calculate the specific growth rate (μ), and this approach successfully identified active microbial populations in digester (positive μ) that could play important roles than those with negative μ. It was further estimated that 82% of microbial populations in the feed sludge were digested in comparison with less than 50% calculated using current equations. PMID:27666090

  3. Identifying hazard parameter to develop quantitative and dynamic hazard map of an active volcano in Indonesia

    Science.gov (United States)

    Suminar, Wulan; Saepuloh, Asep; Meilano, Irwan

    2016-05-01

    Analysis of hazard assessment to active volcanoes is crucial for risk management. The hazard map of volcano provides information to decision makers and communities before, during, and after volcanic crisis. The rapid and accurate hazard assessment, especially to an active volcano is necessary to be developed for better mitigation on the time of volcanic crises in Indonesia. In this paper, we identified the hazard parameters to develop quantitative and dynamic hazard map of an active volcano. The Guntur volcano in Garut Region, West Java, Indonesia was selected as study area due population are resided adjacent to active volcanoes. The development of infrastructures, especially related to tourism at the eastern flank from the Summit, are growing rapidly. The remote sensing and field investigation approaches were used to obtain hazard parameters spatially. We developed a quantitative and dynamic algorithm to map spatially hazard potential of volcano based on index overlay technique. There were identified five volcano hazard parameters based on Landsat 8 and ASTER imageries: volcanic products including pyroclastic fallout, pyroclastic flows, lava and lahar, slope topography, surface brightness temperature, and vegetation density. Following this proposed technique, the hazard parameters were extracted, indexed, and calculated to produce spatial hazard values at and around Guntur Volcano. Based on this method, the hazard potential of low vegetation density is higher than high vegetation density. Furthermore, the slope topography, surface brightness temperature, and fragmental volcanic product such as pyroclastics influenced to the spatial hazard value significantly. Further study to this proposed approach will be aimed for effective and efficient analyses of volcano risk assessment.

  4. Qualitative modeling identifies IL-11 as a novel regulator in maintaining self-renewal in human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Hedi ePeterson

    2013-10-01

    Full Text Available Pluripotency in human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs is regulated by three transcription factors - OCT3/4, SOX2 and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behaviour of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11 and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency.

  5. Bursts of activity in collective cell migration

    CERN Document Server

    Chepizhko, Oleksandr; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stephane; Alava, Mikko J; Zapperi, Stefano; La Porta, Caterina A M

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems.

  6. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells

    Science.gov (United States)

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J.; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-01-01

    Abstract The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication

  7. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Victor Trevino

    2016-04-01

    Full Text Available The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell

  8. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

    Science.gov (United States)

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-04-01

    The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks

  9. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

    Science.gov (United States)

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-04-01

    The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks

  10. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    OpenAIRE

    Segah Altuntaş; Muhammed Ali Kara; Deniz Selin Aksoy; Zehra Dilşad Çoban; Şefik Güran

    2016-01-01

    Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications hav...

  11. An experimental method to identify neurogenic and myogenic active mechanical states of intestinal motility

    Directory of Open Access Journals (Sweden)

    Marcello eCosta

    2013-04-01

    Full Text Available Excitatory and inhibitory enteric neural input to intestinal muscle acting on ongoing myogenic activity determines the rich repertoire of motor patterns involved in digestive function. The enteric neural activity cannot yet be established during movement of intact intestine in vivo or in vitro. We propose the hypothesis that is possible to deduce indirectly, but reliably, the state of activation of the enteric neural input to the muscle from measurements of the mechanical state of the intestinal muscle. The fundamental biomechanical model on which our hypothesis is based is the ‘three-element model’ proposed by Hill. Our strategy is based on simultaneous video recording of changes in diameters and intraluminal pressure with a fibre-optic manometry in isolated segments of rabbit colon. We created a composite spatiotemporal map (DPMap from diameter (DMap and pressure changes (PMaps. In this composite map rhythmic myogenic motor patterns can readily be distinguished from the distension induced neural peristaltic contractions. Plotting the diameter changes against corresponding pressure changes at each location of the segment, generates ‘orbits’ that represent the state of the muscle according to its ability to contract or relax actively or undergoing passive changes. With a software developed in MatLab, we identified twelve possible discrete mechanical states and plotted them showing where the intestine actively contracted and relaxed isometrically, auxotonically or isotonically, as well as where passive changes occurred or was quiescent. Clustering all discrete active contractions and relaxations states generated for the first time a spatio-temporal map of where enteric excitatory and inhibitory neural input to the muscle occurs during physiological movements. Recording internal diameter by an impedance probe proved equivalent to measuring external diameter, making possible to further develop similar strategy in vivo and humans.

  12. Systematic repurposing screening in xenograft models identifies approved drugs with novel anti-cancer activity.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Roix

    Full Text Available Approved drugs target approximately 400 different mechanisms of action, of which as few as 60 are currently used as anti-cancer therapies. Given that on average it takes 10-15 years for a new cancer therapeutic to be approved, and the recent success of drug repurposing for agents such as thalidomide, we hypothesized that effective, safe cancer treatments may be found by testing approved drugs in new therapeutic settings. Here, we report in-vivo testing of a broad compound collection in cancer xenograft models. Using 182 compounds that target 125 unique target mechanisms, we identified 3 drugs that displayed reproducible activity in combination with the chemotherapeutic temozolomide. Candidate drugs appear effective at dose equivalents that exceed current prescription levels, suggesting that additional pre-clinical efforts will be needed before these drugs can be tested for efficacy in clinical trials. In total, we suggest drug repurposing is a relatively resource-intensive method that can identify approved medicines with a narrow margin of anti-cancer activity.

  13. An Active Type I-E CRISPR-Cas System Identified in Streptomyces avermitilis.

    Directory of Open Access Journals (Sweden)

    Yi Qiu

    Full Text Available CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA. The naive features of spacer acquisition in the type I-E system of S. avermitilis were investigated and a completely conserved PAM 5'-AAG-3' was identified. Spacer acquisition displayed differential strand bias upstream and downstream of the priming spacer, and irregular integrations of new spacers were observed. In addition, introduction of this system into host conferred phage resistance to some extent. This study will give new insights into adaptation mechanism of the type I-E systems in vivo, and meanwhile provide theoretical foundation for applying this system on the genetic modification of S. avermitilis.

  14. People who perceive themselves as active cannot identify the intensity recommended by the international physical activity guidelines

    Directory of Open Access Journals (Sweden)

    Prokop NW

    2014-10-01

    Full Text Available Neal W Prokop,1 Travis JR Hrubeniuk,1 Martin Sénéchal,2,3 Danielle R Bouchard1,4 1Faculty of Kinesiology and Recreation Management, University of Manitoba, Winnipeg, Manitoba, Canada; 2Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; 3Department of Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; 4Health, Leisure, and Human Performance Research Institute, Winnipeg, Manitoba, Canada Background: Many national and international organizations recommend that adults achieve at least 150 minutes of aerobic physical activity (PA weekly, at a minimum moderate intensity to optimize health benefits. It is unknown if people who consider themselves as active have the ability to identify what is considered moderate intensity. Methods: Fifty-one participants who reported achieving a minimum 150 minutes per week at a minimum of moderate intensity PA were recruited through a local fitness facility. All participants underwent a single assessment involving questionnaires, clinical measures, and a treadmill test to measure the ability to perceive moderate intensity. Following the visit, participants' PA level was evaluated by heart rate monitor, while exercising, for 7 consecutive days. Results: Eighty percent of participants overestimated moderate intensity on the treadmill test; they were at vigorous intensity compared to what is considered moderate. Only 11.8% of participants accurately identified moderate intensity; all of them were women (P=0.03, had a high level of education (P=0.04, and knew that moderate intensity was the minimum intensity recommended by health organizations (P<0.01. Only 69.2% of participants reached the aerobic component of the International Physical Activity Guidelines with no significant advantage for those correctly identifying moderate intensity. Conclusion: Most people who perceive themselves as active are exercising at vigorous intensity while believing they are

  15. Antibody microarray profiling of osteosarcoma cell serum for identifying potential biomarkers.

    Science.gov (United States)

    Zhu, Zi-Qiang; Tang, Jin-Shan; Gang, Duan; Wang, Ming-Xing; Wang, Jian-Qiang; Lei, Zhou; Feng, Zhou; Fang, Ming-Liang; Yan, Lin

    2015-07-01

    The aim of the present study was to identify biomarkers in osteosarcoma (OS) cell serum by antibody microarray profiling, which may be used for OS diagnosis and therapy. An antibody microarray was used to detect the expression levels of cytokines in serum samples from 20 patients with OS and 20 healthy individuals. Significantly expressed cytokines in OS serum were selected when P2. An enzyme-linked immunosorbent assay (ELISA) was used to validate the antibody microarray results. Finally, classification accuracy was calculated by cluster analysis. Twenty one cytokines were significantly upregulated in OS cell serum samples compared with control samples. Expression of interleukin-6, monocyte chemoattractant protein-1, tumor growth factor-β, growth-related oncogene, hepatocyte growth factor, chemokine ligand 16, Endoglin, matrix metalloproteinase-9 and platelet-derived growth factor-AA was validated by ELISAs. OS serum samples and control samples were distinguished by significantly expressed cytokines with an accuracy of 95%. The results demonstrated that expressed cytokines identified by antibody microarray may be used as biomarkers for OS diagnosis and therapy.

  16. Transcriptome profiling of whole blood cells identifies PLEK2 and C1QB in human melanoma.

    Directory of Open Access Journals (Sweden)

    Yuchun Luo

    Full Text Available Developing analytical methodologies to identify biomarkers in easily accessible body fluids is highly valuable for the early diagnosis and management of cancer patients. Peripheral whole blood is a "nucleic acid-rich" and "inflammatory cell-rich" information reservoir and represents systemic processes altered by the presence of cancer cells.We conducted transcriptome profiling of whole blood cells from melanoma patients. To overcome challenges associated with blood-based transcriptome analysis, we used a PAXgene™ tube and NuGEN Ovation™ globin reduction system. The combined use of these systems in microarray resulted in the identification of 78 unique genes differentially expressed in the blood of melanoma patients. Of these, 68 genes were further analyzed by quantitative reverse transcriptase PCR using blood samples from 45 newly diagnosed melanoma patients (stage I to IV and 50 healthy control individuals. Thirty-nine genes were verified to be differentially expressed in blood samples from melanoma patients. A stepwise logit analysis selected eighteen 2-gene signatures that distinguish melanoma from healthy controls. Of these, a 2-gene signature consisting of PLEK2 and C1QB led to the best result that correctly classified 93.3% melanoma patients and 90% healthy controls. Both genes were upregulated in blood samples of melanoma patients from all stages. Further analysis using blood fractionation showed that CD45(- and CD45(+ populations were responsible for the altered expression levels of PLEK2 and C1QB, respectively.The current study provides the first analysis of whole blood-based transcriptome biomarkers for malignant melanoma. The expression of PLEK2, the strongest gene to classify melanoma patients, in CD45(- subsets illustrates the importance of analyzing whole blood cells for biomarker studies. The study suggests that transcriptome profiling of blood cells could be used for both early detection of melanoma and monitoring of patients

  17. GATA4 knockdown in MA-10 Leydig cells identifies multiple target genes in the steroidogenic pathway.

    Science.gov (United States)

    Bergeron, Francis; Nadeau, Gabriel; Viger, Robert S

    2015-03-01

    GATA4 is an essential transcription factor required for the initiation of genital ridge formation, for normal testicular and ovarian differentiation at the time of sex determination, and for male and female fertility in adulthood. In spite of its crucial roles, the genes and/or gene networks that are ultimately regulated by GATA4 in gonadal tissues remain to be fully understood. This is particularly true for the steroidogenic lineages such as Leydig cells of the testis where many in vitro (promoter) studies have provided good circumstantial evidence that GATA4 is a key regulator of Leydig cell gene expression and steroidogenesis, but formal proof is still lacking. We therefore performed a microarray screening analysis of MA-10 Leydig cells in which Gata4 expression was knocked down using an siRNA strategy. Analysis identified several GATA4-regulated pathways including cholesterol synthesis, cholesterol transport, and especially steroidogenesis. A decrease in GATA4 protein was associated with decreased expression of steroidogenic genes previously suspected to be GATA4 targets such as Cyp11a1 and Star. Gata4 knockdown also led to an important decrease in other novel steroidogenic targets including Srd5a1, Gsta3, Hsd3b1, and Hsd3b6, as well as genes known to participate in cholesterol metabolism such as Scarb1, Ldlr, Soat1, Scap, and Cyp51. Consistent with the decreased expression of these genes, a reduction in GATA4 protein compromised the ability of MA-10 cells to produce steroids both basally and under hormone stimulation. These data therefore provide strong evidence that GATA4 is an essential transcription factor that sits atop of the Leydig cell steroidogenic program. PMID:25504870

  18. A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model

    Energy Technology Data Exchange (ETDEWEB)

    Caruso, Joseph A.; Stemmer, Paul M. [Institute of Environmental Health Sciences, Wayne State University, Detroit, MI (United States); Dombkowski, Alan [Department of Pediatrics, Wayne State University, Detroit, MI (United States); Caruthers, Nicholas J. [Institute of Environmental Health Sciences, Wayne State University, Detroit, MI (United States); Gill, Randall [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States); Rosenspire, Allen J., E-mail: arosenspire@wayne.edu [Department of Immunology and Microbiology, Wayne State University, Detroit, MI (United States)

    2014-04-01

    Network and protein–protein interaction analyses of proteins undergoing Hg{sup 2+}-induced phosphorylation and dephosphorylation in Hg{sup 2+}-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg{sup 2+} exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg{sup 2+}, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg{sup 2+} closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg{sup 2+} led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure. - Highlights: • Inorganic mercury (Hg{sup 2+}) induces changes in the WEHI-231 B cell phosphoproteome. • The B cell receptor (BCR) signaling pathway was the pathway most affected by Hg{sup 2+}. • The Src family phosphoprotein kinase Lyn was the

  19. Identifying components of the hair-cell interactome involved in cochlear amplification

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    Cheatham MaryAnn

    2009-03-01

    Full Text Available Abstract Background Although outer hair cells (OHCs play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23 and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility. Results Using the membrane-based yeast two-hybrid system to screen a newly built cDNA library made predominantly from OHCs, we identified two completely different groups of potential protein partners using prestin and cdh23 as bait. These include both membrane bound and cytoplasmic proteins with 12 being de novo gene products with unknown function(s. In addition, some of these genes are closely associated with deafness loci, implying a potentially important role in hearing. The most abundant prey for prestin (38% is composed of a group of proteins involved in electron transport, which may play a role in OHC survival. The most abundant group of cdh23 prey (55% contains calcium-binding domains. Since calcium performs an important role in hair cell mechanoelectrical transduction and amplification, understanding the interactions between cdh23 and calcium-binding proteins should increase our knowledge of hair cell function at the molecular level. Conclusion The results of this study shed light on some protein networks in cochlear hair cells. Not only was a group of de novo genes closely associated

  20. New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis.

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    Karen G Anthony

    Full Text Available BACKGROUND: In an effort to discover new drugs to treat tuberculosis (TB we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. METHODOLOGY/PRINCIPAL FINDINGS: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. CONCLUSIONS/SIGNIFICANCE: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

  1. Ultrastructural characteristics of novel epithelial cell types identified in human pathologic liver specimens with chronic ductular reaction.

    OpenAIRE

    Vos, R.; Desmet, V

    1992-01-01

    Previous immunohistochemical studies on human liver biopsies with chronic ductular reaction revealed the presence of "small cells" with bile-duct type cytokeratin profile in the periportal area. This study identified similar cells by electron microscopy. The authors studied 13 human liver specimens with various liver diseases, but all characterized by chronic ductular reaction. In all specimens, variable numbers of "small cells" with common epithelial characteristics were identified in the pe...

  2. Chemokines: a new dendritic cell signal for T cell activation

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    Christoph A Thaiss

    2011-08-01

    Full Text Available Dendritic cells (DCs are the main inducers and regulators of cytotoxic T lymphocyte (CTL responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and licensed by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (classical licensing or by NKT cells (alternative licensing. Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

  3. A Clinical Algorithm to Identify HIV Patients at High Risk for Incident Active Tuberculosis: A Prospective 5-Year Cohort Study.

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    Susan Shin-Jung Lee

    Full Text Available Predicting the risk of tuberculosis (TB in people living with HIV (PLHIV using a single test is currently not possible. We aimed to develop and validate a clinical algorithm, using baseline CD4 cell counts, HIV viral load (pVL, and interferon-gamma release assay (IGRA, to identify PLHIV who are at high risk for incident active TB in low-to-moderate TB burden settings where highly active antiretroviral therapy (HAART is routinely provided.A prospective, 5-year, cohort study of adult PLHIV was conducted from 2006 to 2012 in two hospitals in Taiwan. HAART was initiated based on contemporary guidelines (CD4 count < = 350/μL. Cox regression was used to identify the predictors of active TB and to construct the algorithm. The validation cohorts included 1455 HIV-infected individuals from previous published studies. Area under the receiver operating characteristic (ROC curve was calculated.Seventeen of 772 participants developed active TB during a median follow-up period of 5.21 years. Baseline CD4 < 350/μL or pVL ≥ 100,000/mL was a predictor of active TB (adjusted HR 4.87, 95% CI 1.49-15.90, P = 0.009. A positive baseline IGRA predicted TB in patients with baseline CD4 ≥ 350/μL and pVL < 100,000/mL (adjusted HR 6.09, 95% CI 1.52-24.40, P = 0.01. Compared with an IGRA-alone strategy, the algorithm improved the sensitivity from 37.5% to 76.5%, the negative predictive value from 98.5% to 99.2%. Compared with an untargeted strategy, the algorithm spared 468 (60.6% from unnecessary TB preventive treatment. Area under the ROC curve was 0.692 (95% CI: 0.587-0.798 for the study cohort and 0.792 (95% CI: 0.776-0.808 and 0.766 in the 2 validation cohorts.A validated algorithm incorporating the baseline CD4 cell count, HIV viral load, and IGRA status can be used to guide targeted TB preventive treatment in PLHIV in low-to-moderate TB burden settings where HAART is routinely provided to all PLHIV. The implementation of this algorithm will avoid unnecessary

  4. Entangled active matter: From cells to ants

    Science.gov (United States)

    Hu, D. L.; Phonekeo, S.; Altshuler, E.; Brochard-Wyart, F.

    2016-07-01

    Both cells and ants belong to the broad field of active matter, a novel class of non-equilibrium materials composed of many interacting units that individually consume energy and collectively generate motion or mechanical stresses. However cells and ants differ from fish and birds in that they can support static loads. This is because cells and ants can be entangled, so that individual units are bound by transient links. Entanglement gives cells and ants a set of remarkable properties usually not found together, such as the ability to flow like a fluid, spring back like an elastic solid, and self-heal. In this review, we present the biology, mechanics and dynamics of both entangled cells and ants. We apply concepts from soft matter physics and wetting to characterize these systems as well as to point out their differences, which arise from their differences in size. We hope that our viewpoints will spur further investigations into cells and ants as active materials, and inspire the fabrication of synthetic active matter.

  5. Negative regulation of active zone assembly by a newly identified SR protein kinase.

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    Ervin L Johnson

    2009-09-01

    Full Text Available Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila or ribbon (vertebrates are believed to facilitate vesicle movement to the active zone (AZ of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D. This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  6. Using Light-at-Night (LAN) Satellite Data for Identifying Clusters of Economic Activities in Europe

    Science.gov (United States)

    Rybnikova, N. A.; Portnov, B. A.

    2015-04-01

    Enterprises organized in clusters are often efficient in stimulating urban development, productivity and profit outflows. Identifying clusters of economic activities (EAs) thus becomes an important step in devising regional development policies, aimed at facilitating regional economic development. However, a major problem with cluster identification stems from limited reporting of specific EAs by individual countries and administrative entities. Even Eurostat, which maintains most advances regional databases, provides data for less than 50% of all regional subdivisions of the 3rd tier of the Nomenclature of Territorial Units for Statistics (NUTS3). Such poor reporting impedes identification of EA clusters and economic forces behind them. In this study, we test a possibility that missing data on geographic concentrations of EAs can be reconstructed using Light-at-Night (LAN) satellite measurements, and that such reconstructed data can then be used for the identification of EA clusters. As we hypothesize, LAN, captured by satellite sensors, is characterized by different intensity, depending on its source - production facilities, services, etc., - and this information can be used for EA identification. The study was carried out in three stages. First, using nighttime satellite images, we determined what types of EAs can be identified, with a sufficient degree of accuracy, by LAN they emit. Second, we calculated multivariate statistical models, linking EAs concentrations with LAN intensities and several locational and development attributes of NUTS3 regions in Europe. Next, using the obtained statistical models, we restored missing data on EAs across NUTS3 regions in Europe and identified clusters of EAs, using spatial analysis tools.

  7. Identifying Feasible Physical Activity Programs for Long-Term Care Homes in the Ontario Context

    Science.gov (United States)

    Shakeel, Saad; Newhouse, Ian; Malik, Ali; Heckman, George

    2015-01-01

    Background Structured exercise programs for frail institutionalized seniors have shown improvement in physical, functional, and psychological health of this population. However, the ‘feasibility’ of implementation of such programs in real settings is seldom discussed. The purpose of this systematic review was to gauge feasibility of exercise and falls prevention programs from the perspective of long-term care homes in Ontario, given the recent changes in funding for publically funded physiotherapy services. Method Six electronic databases were searched by two independent researchers for randomized controlled trials that targeted long-term care residents and included exercise as an independent component of the intervention. Results A total of 39 studies were included in this review. A majority of these interventions were led by physiotherapist(s), carried out three times per week for 30–45 minutes per session. However, a few group-based interventions that were led by long-term care staff, volunteers, or trained non-exercise specialists were identified that also required minimal equipment. Conclusion This systematic review has identified ‘feasible’ physical activity and falls prevention programs that required minimal investment in staff and equipment, and demonstrated positive outcomes. Implementation of such programs represents cost-effective means of providing long-term care residents with meaningful gains in physical, psychological, and social health. PMID:26180563

  8. Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli.

    Science.gov (United States)

    Maher, Marie C; Lim, Ji Youn; Gunawan, Cheston; Cegelski, Lynette

    2015-10-01

    Escherichia coli assemble functional amyloid fibers termed curli that contribute to bacterial adhesion, biofilm formation, and host pathogenesis. We developed a cell-based high-throughput screen to identify inhibitors of curli-mediated adhesion in the laboratory strain MC4100 and curli-associated biofilm formation in the uropathogenic E. coli clinical isolate UTI89. Inhibitors of biofilm formation can operate through many mechanisms, and such inhibitors could hold therapeutic value in preventing and treating urinary tract infections. The curli-specific screen allows the identification of compounds that inhibit either curli expression, curli biogenesis, or adhesion by normally produced curli. In screening the NIH Clinical Collection of 446 compounds, we identified rifapentine as a potent inhibitor in both of these screens. Rifapentine is an antibiotic used to treat tuberculosis that targets RNA polymerase, but prevents curli-dependent adhesion and biofilm formation in E. coli at concentrations below those that affect viability. Rifapentine inhibits curli production and prevents biofilm formation on plastic, on agar, and at the air-liquid interface by inhibiting curli gene transcription. Comparisons with a cephalosporin antibiotic further revealed that curli production is not affected by standard antibiotic treatment and cell killing pressure. Thus, we reveal a new role independent of killing activity for rifapentine as an inhibitor of curli and curli-mediated biofilm formation. PMID:27623312

  9. Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

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    Steve Pells

    Full Text Available Human embryonic stem cells (hESCs undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency "signature" would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5 induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

  10. Critical telomerase activity for uncontrolled cell growth.

    Science.gov (United States)

    Wesch, Neil L; Burlock, Laura J; Gooding, Robert J

    2016-01-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed. PMID:27500377

  11. Critical telomerase activity for uncontrolled cell growth

    Science.gov (United States)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  12. Raman spectroscopy identifies radiation response in human non-small cell lung cancer xenografts

    Science.gov (United States)

    Harder, Samantha J.; Isabelle, Martin; Devorkin, Lindsay; Smazynski, Julian; Beckham, Wayne; Brolo, Alexandre G.; Lum, Julian J.; Jirasek, Andrew

    2016-02-01

    External beam radiation therapy is a standard form of treatment for numerous cancers. Despite this, there are no approved methods to account for patient specific radiation sensitivity. In this report, Raman spectroscopy (RS) was used to identify radiation-induced biochemical changes in human non-small cell lung cancer xenografts. Chemometric analysis revealed unique radiation-related Raman signatures that were specific to nucleic acid, lipid, protein and carbohydrate spectral features. Among these changes was a dramatic shift in the accumulation of glycogen spectral bands for doses of 5 or 15 Gy when compared to unirradiated tumours. When spatial mapping was applied in this analysis there was considerable variability as we found substantial intra- and inter-tumour heterogeneity in the distribution of glycogen and other RS spectral features. Collectively, these data provide unique insight into the biochemical response of tumours, irradiated in vivo, and demonstrate the utility of RS for detecting distinct radiobiological responses in human tumour xenografts.

  13. Application of laser microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells.

    Science.gov (United States)

    Berruti, Andrea; Borriello, Roberto; Lumini, Erica; Scariot, Valentina; Bianciotto, Valeria; Balestrini, Raffaella

    2013-01-01

    Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum) are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intra-cellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells (AC) through a laser microdissection (LMD) approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the AC. Unexpectedly, one taxon was found in the AC, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community.

  14. Chemical Library Screening and Structure-Function Relationship Studies Identify Bisacodyl as a Potent and Selective Cytotoxic Agent Towards Quiescent Human Glioblastoma Tumor Stem-Like Cells.

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    Maria Zeniou

    Full Text Available Cancer stem-like cells reside in hypoxic and slightly acidic tumor niches. Such microenvironments favor more aggressive undifferentiated phenotypes and a slow growing "quiescent state" which preserves them from chemotherapeutic agents that essentially target proliferating cells. Our objective was to identify compounds active on glioblastoma stem-like cells, including under conditions that mimick those found in vivo within this most severe and incurable form of brain malignancy. We screened the Prestwick Library to identify cytotoxic compounds towards glioblastoma stem-like cells, either in a proliferating state or in more slow-growing "quiescent" phenotype resulting from non-renewal of the culture medium in vitro. Compound effects were assessed by ATP-level determination using a cell-based assay. Twenty active molecules belonging to different pharmacological classes have thus been identified. Among those, the stimulant laxative drug bisacodyl was the sole to inhibit in a potent and specific manner the survival of quiescent glioblastoma stem-like cells. Subsequent structure-function relationship studies led to identification of 4,4'-dihydroxydiphenyl-2-pyridyl-methane (DDPM, the deacetylated form of bisacodyl, as the pharmacophore. To our knowledge, bisacodyl is currently the only known compound targeting glioblastoma cancer stem-like cells in their quiescent, more resistant state. Due to its known non-toxicity in humans, bisacodyl appears as a new potential anti-tumor agent that may, in association with classical chemotherapeutic compounds, participate in tumor eradication.

  15. Targeting of MAPK-associated molecules identifies SON as a prime target to attenuate the proliferation and tumorigenicity of pancreatic cancer cells

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    Furukawa Toru

    2012-12-01

    Full Text Available Abstract Background Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinase (MAPK. Activation of MAPK is associated with the upregulation of genes implicated in the proliferation and survival of pancreatic cancer cells. We hypothesized that knockdown of these MAPK-associated molecules could produce notable anticancer phenotypes. Methods A RNA interference-mediated knockdown screening of 78 MAPK-associated molecules previously identified was performed to find molecules specifically associated with proliferation of pancreatic cancer cells in vitro. Expression of an identified molecule in pancreatic cancer tissues was examined by immunohistochemistry. In vivo tumorigenicity of cancer cells with stable knockdown of the molecule was assayed by using xenograft models. Flow cytometry and live cell imaging were employed to assess an association of the molecule with cell cycle. Results The knockdown screening revealed that knockdown of SON, the gene encoding SON, which is a large serine/arginine-rich protein involved in RNA processing, substantially suppressed pancreatic cancer cell proliferation and survival in vitro and tumorigenicity in vivo. SON expression was higher in ductal adenocarcinomas than in cells of normal ducts and precursor lesions in pancreatic cancer tissues. Knockdown of SON induced G2/M arrest and apoptosis in cultured cancer cells. The suppressive effect of SON knockdown on proliferation was less pronounced in cultured normal duct epithelial cells. SON formed nuclear speckles in the interphase of the cell cycle and dispersed in the cytoplasm during mitosis. Live cell imaging showed that SON diffusely dispersed in the early mitotic phase, accumulated in some foci in the cytoplasm in the late mitotic phase, and gradually reassembled into speckles after mitosis. Conclusion These results indicate that SON plays a critical role in the proliferation, survival, and tumorigenicity of pancreatic

  16. Circulating Tumor Cells: A Review of Present Methods and the Need to Identify Heterogeneous Phenotypes

    Science.gov (United States)

    Millner, Lori M.; Linder, Mark W.; Valdes, Roland

    2016-01-01

    The measurement and characterization of circulating tumor cells (CTCs) hold promise for advancing personalized therapeutics. CTCs are the precursor to metastatic cancer and thus have the potential to radically alter patient treatment and outcome. Currently, clinical information provided by the enumeration of CTCs is limited to predicting clinical outcome. Other areas of interest in advancing the practice of pathology include: using CTCs for early detection of potential metastasis, determining and monitoring the efficacy of individualized treatment regimens, and predicting site-specific metastasis. Important hurdles to overcome in obtaining this type of clinical information involve present limitations in defining, detecting, and isolating CTCs. Currently, CTCs are detected using epithelial markers. The definition of what distinguishes a CTC should be expanded to include CTCs with heterogeneous phenotypes, and markers should be identified to enable a more comprehensive capture. Additionally, most methods available for detecting CTCs do not capture functionally viable CTCs. Retaining functional viability would provide a significant advantage in characterizing CTC-subtypes that may predict the site of metastatic invasion and thus assist in selecting effective treatment regimens. In this review we describe areas of clinical interest followed by a summary of current circulating cell-separation technologies and present limitations. Lastly, we provide insight into what is required to overcome these limitations as they relate to applications in advancing the practice of pathology and laboratory medicine. PMID:23884225

  17. Deletion of Scap in alveolar type II cells influences lung lipid homeostasis and identifies a compensatory role for pulmonary lipofibroblasts.

    Science.gov (United States)

    Besnard, Valérie; Wert, Susan E; Stahlman, Mildred T; Postle, Anthony D; Xu, Yan; Ikegami, Machiko; Whitsett, Jeffrey A

    2009-02-01

    Pulmonary function after birth is dependent upon surfactant lipids that reduce surface tension in the alveoli. The sterol-responsive element-binding proteins (SREBPs) are transcription factors regulating expression of genes controlling lipid homeostasis in many tissues. To identify the role of SREBPs in the lung, we conditionally deleted the SREBP cleavage-activating protein gene, Scap, in respiratory epithelial cells (ScapDelta/Delta) in vivo. Prior to birth (E18.5), deletion of Scap decreased the expression of both SREBPs and a number of genes regulating fatty acid and cholesterol metabolism. Nevertheless, ScapDelta/Delta mice survived postnatally, surfactant and lung tissue lipids being substantially normalized in adult ScapDelta/Delta mice. Although phospholipid synthesis was decreased in type II cells from adult ScapDelta/Delta mice, lipid storage, synthesis, and transfer by lung lipofibroblasts were increased. mRNA microarray data indicated that SCAP influenced two major gene networks, one regulating lipid metabolism and the other stress-related responses. Deletion of the SCAP/SREBP pathway in respiratory epithelial cells altered lung lipid homeostasis and induced compensatory lipid accumulation and synthesis in lung lipofibroblasts. PMID:19074148

  18. New Alzheimer amyloid beta responsive genes identified in human neuroblastoma cells by hierarchical clustering.

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    Markus Uhrig

    Full Text Available Alzheimer's disease (AD is characterized by neuronal degeneration and cell loss. Abeta(42, in contrast to Abeta(40, is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Abeta(40 and Abeta(42 levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Abeta(40 and Abeta(42 levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2 and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Abeta(42/Abeta(40 ratio. Importantly however, an increased Abeta(42/Abeta(40 ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Abeta(42/Abeta(40 ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Abeta(42/Abeta(40 ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes.

  19. New Alzheimer Amyloid β Responsive Genes Identified in Human Neuroblastoma Cells by Hierarchical Clustering

    Science.gov (United States)

    Uhrig, Markus; Ittrich, Carina; Wiedmann, Verena; Knyazev, Yuri; Weninger, Annette; Riemenschneider, Matthias; Hartmann, Tobias

    2009-01-01

    Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Aβ42, in contrast to Aβ40, is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Aβ40 and Aβ42 levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Aβ40 and Aβ42 levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Aβ42/Aβ40 ratio. Importantly however, an increased Aβ42/Aβ40 ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Aβ42/Aβ40 ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Aβ42/Aβ40 ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes. PMID:19707560

  20. New Alzheimer amyloid beta responsive genes identified in human neuroblastoma cells by hierarchical clustering.

    Science.gov (United States)

    Uhrig, Markus; Ittrich, Carina; Wiedmann, Verena; Knyazev, Yuri; Weninger, Annette; Riemenschneider, Matthias; Hartmann, Tobias

    2009-01-01

    Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Abeta(42), in contrast to Abeta(40), is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Abeta(40) and Abeta(42) levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Abeta(40) and Abeta(42) levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Abeta(42)/Abeta(40) ratio. Importantly however, an increased Abeta(42)/Abeta(40) ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Abeta(42)/Abeta(40) ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Abeta(42)/Abeta(40) ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes. PMID:19707560

  1. Mechanically activated artificial cell by using microfluidics.

    Science.gov (United States)

    Ho, Kenneth K Y; Lee, Lap Man; Liu, Allen P

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

  2. Mechanically activated artificial cell by using microfluidics

    Science.gov (United States)

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  3. Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.

    Science.gov (United States)

    Fokkelman, Michiel; Balcıoğlu, Hayri E; Klip, Janna E; Yan, Kuan; Verbeek, Fons J; Danen, Erik H J; van de Water, Bob

    2016-01-01

    Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour. PMID:27531518

  4. WANO Activities Related to Identifying and Reducing the Likelihood for Recurring Events

    International Nuclear Information System (INIS)

    Since its inception, WANO has encouraged members to share operating experience and events information through the WANO Operating Experience Programme. Preventing recurring events is a prime reason for sharing events information. Over 2500 events have been shared through WANO since 1989. However, a review of WANO activities in 1997 identified that this information was not being used very well by WANO members, and that WANO was not adding much 'value' to the events sharing process. At the time, WANO only provided the 'postal exchange' function for events sharing, and was not reviewing events across WANO to help members focus on the really important issues. Often, these very important issues involve recurring events. As a result of the 1997 review of the WANO operating experience process, WANO re-sourced and developed new analysis capabilities and began producing new types of reports for its members. The resource commitment includes four seconded engineers (one from each WANO Regional Centre) brought to Paris to staff a WANO Operating Experience Central Team. This team analyses events across WANO, writes WANO event reports, provides operating experience-related training, and provides technical support to members to improve their use of operating experience information. One focus area for events analysis by WANO is the identification and subsequent industry notification of recurring events. As a result of the 1997 review of WANO activities, in 1998 WANO began production of several new types of event-based reports to communicate significant industry events to our members. A key focus of analysis of these significant events is whether they are recurring events (that is, very similar to previous events either at that NPP or another NPP in the industry). The reports are called Significant Operating Experience Reports (SOERs) and Significant Event Reports (SERs). Significant Operating Experience Reports (SOERs) are written by WANO when several event reports indicate that

  5. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

    Directory of Open Access Journals (Sweden)

    Timothy M Butler

    Full Text Available The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  6. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    Science.gov (United States)

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Gray, Joe W.; Spellman, Paul T.

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor. PMID:26317216

  7. Tomato waste: Carotenoids content, antioxidant and cell growth activities.

    Science.gov (United States)

    Stajčić, Sladjana; Ćetković, Gordana; Čanadanović-Brunet, Jasna; Djilas, Sonja; Mandić, Anamarija; Četojević-Simin, Dragana

    2015-04-01

    The carotenoid content, antioxidant and cell growth activities of tomato waste extracts, obtained from five different tomato genotypes, was investigated. High performance liquid chromatography was used to identify and quantify the main carotenoids present in tomato waste extracts. The antioxidant activity of tomato waste extracts was tested using spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power assay. The highest DPPH scavenging activity (IC50 = 0.057 mg/ml) was obtained for Bačka extract. The Knjaz extract showed the best reducing power (IC50 = 2.12 mg/ml). Cell growth effects were determined in HeLa, MCF7 and MRC-5 cell lines by sulforhodamine B test. Anti-proliferative effects were observed in all cell lines at higher concentrations (⩾ 0.125 mg/ml). The carotenoid contents exhibited a strong correlation with antioxidant and anti-proliferation activity. The results obtained indicated that tomato waste should be regarded as potential nutraceutic resource and may be used as a functional food ingredient. PMID:25442547

  8. Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Elizabeth Bilsland

    2016-01-01

    Full Text Available Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7-15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts, and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds, we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident

  9. p16(INK4a) /Ki-67 co-expression specifically identifies transformed cells in the head and neck region.

    Science.gov (United States)

    Prigge, Elena-Sophie; Toth, Csaba; Dyckhoff, Gerhard; Wagner, Steffen; Müller, Franziska; Wittekindt, Claus; Freier, Kolja; Plinkert, Peter; Hoffmann, Jürgen; Vinokurova, Svetlana; Klussmann, Jens Peter; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2015-04-01

    p16(INK4a) immunohistochemical overexpression is an overall reliable surrogate marker of human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCC). However, cases of ambiguous p16(INK4a) overexpression are regularly detected in the head and neck: p16(INK4a) expression can be observed in non-malignant tissue, such as tonsillar crypt epithelium and a proportion of branchial cleft cysts. Additionally, diverse patterns of p16(INK4) expression can complicate interpretation of "p16(INK4a) -positivity". These aspects impede the unrestricted application of p16(INK4a) as a diagnostic marker in the head and neck. We hypothesized that combined detection of p16(INK4a) and the proliferation marker Ki-67 could support clarification of ambiguous p16(INK4a) expression in the head and neck by specifically indicating p16(INK4a) -expressing cells with proliferative activity. p16(INK4a) /Ki-67 co-expression in a combined staining procedure was correlated to distinct p16(INK4a) expression patterns and HPV status (HPV DNA followed by E6*I oncogene mRNA detection) in 147 HNSCC and 50 non-malignant head and neck samples. p16(INK4a) /Ki-67 co-expression only occurred in transformed cells of the head and neck. Co-expression was never detected in non-transformed cells. Combined p16(INK4a) /Ki-67 expression was stringently associated with a diffuse p16(INK4a) expression pattern. All HPV oncogene-expressing HNSCC showed p16(INK4a) /Ki-67 co-expression. We demonstrate that p16(INK4a) /Ki-67 co-expression occurs exclusively in transformed cells of the head and neck. Our findings indicate a substantial impact of combined p16(INK4a) /Ki-67 expression in the assessment of ambiguous p16(INK4a) expression in the head and neck by specifically identifying p16(INK4a) -expressing cells with proliferative activity. This property will be of considerable significance for head and neck histo- and cytopathology.

  10. Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity.

    Science.gov (United States)

    Lee, Caroline A; Jones, J P; Katayama, Jonathan; Kaspera, Rüdiger; Jiang, Ying; Freiwald, Sascha; Smith, Evan; Walker, Gregory S; Totah, Rheem A

    2012-05-01

    CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues. PMID:22328583

  11. A yeast surface display system for the discovery of ligands that trigger cell activation.

    Science.gov (United States)

    Cho, B K; Kieke, M C; Boder, E T; Wittrup, K D; Kranz, D M

    1998-11-01

    Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

  12. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals.

    Science.gov (United States)

    Achkar, Jacqueline M; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-09-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  13. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    Science.gov (United States)

    Achkar, Jacqueline M.; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O.; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  14. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  15. Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

    Science.gov (United States)

    Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A; Ballok, Alicia E; Bomberger, Jennifer M; Cady, Kyle C; O'Toole, George A; Madden, Dean R

    2014-03-14

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections. PMID:24474692

  16. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Directory of Open Access Journals (Sweden)

    Guan-Feng Wang

    2015-02-01

    Full Text Available Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR. Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC, a nucleotide binding (NB-ARC and a leucine rich repeat (LRR domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  17. Full field optical coherence tomography can identify spermatogenesis in a rodent sertoli-cell only model

    Directory of Open Access Journals (Sweden)

    Ranjith Ramasamy

    2012-01-01

    Full Text Available Background: Microdissection testicular sperm extraction (micro-TESE has replaced conventional testis biopsies as a method of choice for obtaining sperm for in vitro fertilization for men with nonobstructive azoospermia. A technical challenge of micro-TESE is that the low magnification inspection of the tubules with a surgical microscope is insufficient to definitively identify sperm-containing tubules, necessitating tissue removal and cytologic assessment. Full field optical coherence tomography (FFOCT uses white light interference microscopy to generate quick high-resolution tomographic images of fresh (unprocessed and unstained tissue. Furthermore, by using a nonlaser safe light source (150 W halogen lamp for tissue illumination, it ensures that the sperm extracted for in vitro fertilization are not photo-damaged or mutagenized. Materials and Methods: A focal Sertoli-cell only rodent model was created with busulfan injection in adult rats. Ex vivo testicular tissues from both normal and busulfan-treated rats were imaged with a commercial modified FFOCT system, Light-CT TM , and the images were correlated with gold standard hematoxylin and eosin staining. Results: Light-CT TM identified spermatogenesis within the seminiferous tubules in freshly excised testicular tissue, without the use of exogenous contrast or fixation. Normal adult rats exhibited tubules with uniform size and shape (diameter 328 ±11 μm. The busulfan-treated animals showed marked heterogeneity in tubular size and shape (diameter 178 ± 35 μm and only 10% contained sperm within the lumen. Conclusion : FFOCT has the potential to facilitate real-time visualization of spermatogenesis in humans, and aid in micro-TESE for men with infertility.

  18. Activation-Induced Cell Death in T Cells and Autoimmunity

    Institute of Scientific and Technical Information of China (English)

    Jian Zhang; Xuemei Xu; Yong Liu

    2004-01-01

    Activation-induced cell death (AICD), which results from the interaction between Fas and Fas ligand, is responsible for maintaining tolerance to self-antigen. A defect in AICD may lead to development of autoimmunity. During the last several years, much progress has been made in understanding the mechanism(s) of AICD and its potential role in the pathogenesis of autoimmune diseases. In this review, we summarize the most recent progress on the regulation of the susceptibility of T cells to AICD and its possible involvement in autoimmune diseases.

  19. Integrin α6β4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease.

    Directory of Open Access Journals (Sweden)

    Xiaopeng Li

    Full Text Available To develop stem/progenitor cell-based therapy for cystic fibrosis (CF lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4(+ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4(+ epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4(- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4(+ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs serotypes, AAV2 and AAV8, capable of transducing α6β4(+ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4(+ epithelial cells significantly rescued defects in Cl(- transport. Therefore, targeting the α6β4(+ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

  20. Decidual cell polyploidization necessitates mitochondrial activity.

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    Xinghong Ma

    Full Text Available Cellular polyploidy has been widely reported in nature, yet its developmental mechanism and function remain poorly understood. In the present study, to better define the aspects of decidual cell polyploidy, we isolated pure polyploid and non-polyploid decidual cell populations from the in vivo decidual bed. Three independent RNA pools prepared for each population were then subjected to the Affymetrix gene chip analysis for the whole mouse genome transcripts. Our data revealed up-regulation of 1015 genes and down-regulation of 1207 genes in the polyploid populations, as compared to the non-polyploid group. Comparative RT-PCR and in situ hybridization results indeed confirmed differential expressional regulation of several genes between the two populations. Based on functional enrichment analyses, up-regulated polyploidy genes appeared to implicate several functions, which primarily include cell/nuclear division, ATP binding, metabolic process, and mitochondrial activity, whereas that of down-regulated genes primarily included apoptosis and immune processes. Further analyses of genes that are related to mitochondria and bi-nucleation showed differential and regional expression within the decidual bed, consistent with the pattern of polyploidy. Consistently, studies revealed a marked induction of mitochondrial mass and ATP production in polyploid cells. The inhibition of mitochondrial activity by various pharmacological inhibitors, as well as by gene-specific targeting using siRNA-mediated technology showed a dramatic attenuation of polyploidy and bi-nucleation development during in vitro stromal cell decidualization, suggesting mitochondria play a major role in positive regulation of decidual cell polyploidization. Collectively, analyses of unique polyploidy markers and molecular signaling networks may be useful to further characterize functional aspects of decidual cell polyploidy at the site of implantation.

  1. Identifying different regimes in eruptive activity: An application to Etna volcano

    Science.gov (United States)

    Mulargia, F.; Gasperini, P.; Tinti, S.

    1987-12-01

    The objective identification of different regimes in the eruptive time-history of a volcano is crucial to the understanding of its physics. While a problem well-known in statistical literature under the name of change-point or scan-point problem, no method of general applicability exists for the identification of different regimes in a time-series. In particular, the available techniques seem unsuitable to the volcanological case. We developed an original procedure based on two-sample Kolmogorov-Smirnov statistics which offers satisfactory accuracy in a broad range of conditions with a minimum of assumptions and is expressly tailored to the study of geophysical phenomena. Our procedure requires neither the a priori knowledge of the number of regimes nor of the statistical distributions governing the whole process, which can be of different type. The parent distribution of each regime is inferred through a goodness-of-fit test, and this in turn allows the confidence intervals for each of the change-points identified to be estimated by numerical simulation. This procedure is applied to the eruptive history of Mount Etna volcano. Available data allow the analysis of flank eruptions in the period 1600-1980, while the total output (summit and flank activity) can be studied only in the period 1971-1981. Information on eruptive history can be therefore obtained at two different timescales. Since no univocally accepted catalog exists except for the last few decades, we use two different sets of data, which practically exhaust all the available information. The results are interpreted by a stability analysis, and only stable results are retained. Our analysis yields that: - The inter-event times of flank eruptions in the period 1600-1980 follow two regimes before and after year 1865, while the eruptive activity in the period 1971-1981 follows four different regimes. In each regime eruptions occur according to a Poisson process and Etna behaves as a random nonstationary

  2. Restoring KLF5 in esophageal squamous cell cancer cells activates the JNK pathway leading to apoptosis and reduced cell survival.

    Science.gov (United States)

    Tarapore, Rohinton S; Yang, Yizeng; Katz, Jonathan P

    2013-05-01

    Esophageal cancer is the eighth most common cancer in the world and has an extremely dismal prognosis, with a 5-year survival of less than 20%. Current treatment options are limited, and thus identifying new molecular targets and pathways is critical to derive novel therapies. Worldwide, more than 90% of esophageal cancers are esophageal squamous cell cancer (ESCC). Previously, we identified that Krüppel-like factor 5 (KLF5), a key transcriptional regulator normally expressed in esophageal squamous epithelial cells, is lost in human ESCC. To examine the effects of restoring KLF5 in ESCC, we transduced the human ESCC cell lines TE7 and TE15, both of which lack KLF5 expression, with retrovirus to express KLF5 upon doxycycline induction. When KLF5 was induced, ESCC cells demonstrated increased apoptosis and decreased viability, with up-regulation of the proapoptotic factor BAX. Interestingly, c-Jun N-terminal kinase (JNK) signaling, an important upstream mediator of proapoptotic pathways including BAX, was also activated following KLF5 induction. KLF5 activation of JNK signaling was mediated by KLF5 transactivation of two key upstream regulators of the JNK pathway, ASK1 and MKK4, and inhibition of JNK blocked apoptosis and normalized cell survival following KLF5 induction. Thus, restoring KLF5 in ESCC cells promotes apoptosis and decreases cell survival in a JNK-dependent manner, providing a potential therapeutic target for human ESCC.

  3. Electrodynamic activity of healthy and cancer cells

    International Nuclear Information System (INIS)

    Microtubules in the cell form a structure capable of generating electrodynamic field and mitochondria form their supporting system for physical processes including energy supply. Mitochondria transfer protons from their matrix space into cytosol, create strong static field around them that causes ordering of water and altering it into quasi-elastic medium with reduced viscous damping. Microtubules are composed of heterodimers that are electric dipoles. Microtubule oscillations generate an electrodynamic field. The greatest energy supply may be provided by liberation of non-utilized energy from mitochondria. Microtubules and mitochondria form a unique cooperating system in the cell. Mitochondria form a boundary element whose function depends on chemical-genetic control but their output is essential for physical processes in the cell. Mitochondrial dysfunction in cancer cells results in diminished intensity of the static electric field, disturbed water ordering, increased damping of microtubule oscillations and their shift towards linear region, and decreased energy supply. Power and coherence of oscillations and generated electrodynamic field is weakened. Malignant properties of cancer cell, in particular local invasion and metastasis, may depend on disturbed electrodynamic field. Nanotechnology is promising for investigation of electrodynamic activity in living cells.

  4. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse;

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19......) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein...... expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding...

  5. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    Science.gov (United States)

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  6. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila.

    Science.gov (United States)

    Liu, Ying; Ge, Qinglan; Chan, Brian; Liu, Hanhan; Singh, Shree Ram; Manley, Jacob; Lee, Jae; Weideman, Ann Marie; Hou, Gerald; Hou, Steven X

    2016-01-01

    Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level. PMID:27484291

  7. Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells

    Directory of Open Access Journals (Sweden)

    Wu Jian

    2012-08-01

    Full Text Available Abstract Background Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37. Results Ten compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide and late apoptotic cells (Annexin V+/PI+ increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively. Conclusions Colchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells.

  8. Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

    Directory of Open Access Journals (Sweden)

    Simon R Stockwell

    Full Text Available Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical

  9. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  10. Identifying the controls of wildfire activity in Namibia using multivariate statistics

    Science.gov (United States)

    Mayr, Manuel; Le Roux, Johan; Samimi, Cyrus

    2015-04-01

    Despite large areas of Namibia being unaffected by fires due to aridity, substantial burning in the northern and north-eastern parts of the country is observed every year. Within the fire-affected regions, a strong spatial and inter-annual variability characterizes the dry-season fire situation. In order to understand these patterns, it appears critical to identify the causative factors behind fire occurrence and to examine their interactions in detail. Furthermore, most studies dealing with causative factor examination focus either on the local or the regional scale. However, these scales seem to be inappropriate from a management perspective, as fire-related strategic action plans are most often set up nationwide. Here, we will present an examination of the fire regimes of Namibia based on a dataset conducted by Le Roux (2011). A decade-spanning fire record (1994-2003) derived from NOAA's Advanced Very High Resolution Radiometer (AVHRR) imagery was used to generate four fire regime metrics (Burned Area, Fire Season Length, Month of Peak Fire Season, and Fire Return Period) and quantitative information on vegetation and phenology derived from Normalized Difference Vegetation Index (NDVI) time series. Further variables contained by this dataset are related to climate, biodiversity, and human activities. Le Roux (2011) analyzed the correlations between the fire metrics mentioned above and the predictor variables. We hypothesize that linear correlations (as estimated by correlation coefficients) simplify the interactions between response and predictor variables. For instance, moderate population densities could induce the highest number of fires, whereas the complete absence of humans lacks one major source of ignition. Around highly populated areas, in contrary, fuels are usually reduced and space is more fragmented - thus, the initiation and spread of a potential fire could as well be inhibited. From a total of over 40 explanatory variables, we will initially use

  11. CD62Lneg CD38+ expression on circulating CD4 + T cells identifies mucosally differentiated cells in protein fed mice and in human celiac disease patients and controls

    NARCIS (Netherlands)

    M.F. du Pré (Fleur); L.A. van Berkel (Lisette); M. Ráki (Melinda); M.A. Van Leeuwen (Marieke); L.F. de Ruiter (Lilian); F. Broere; M.N.D. Ter Borg (Mariëtte N. D.); F.E. Lund (Frances E.); J.C. Escher (Johanna); K.E.A. Lundin (Knut E. A.); L.M. Sollid (Ludvig M.); G. Kraal; E.E.S. Nieuwenhuis (Edward); J.N. Samsom (Janneke)

    2011-01-01

    textabstractObjectives: The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood. Methods: Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph no

  12. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

    Science.gov (United States)

    Dalton, Jutta C; Bätz, Ulrike; Liu, Jason; Curie, Gemma L; Quail, Peter H

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation. PMID:27379152

  13. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

    Science.gov (United States)

    Dalton, Jutta C; Bätz, Ulrike; Liu, Jason; Curie, Gemma L; Quail, Peter H

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation.

  14. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3

    Science.gov (United States)

    Dalton, Jutta C.; Bätz, Ulrike; Liu, Jason; Curie, Gemma L.; Quail, Peter H.

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5′-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation. PMID:27379152

  15. IDENTIFYING LUMINOUS ACTIVE GALACTIC NUCLEI IN DEEP SURVEYS: REVISED IRAC SELECTION CRITERIA

    International Nuclear Information System (INIS)

    Spitzer/IRAC selection is a powerful tool for identifying luminous active galactic nuclei (AGNs). For deep IRAC data, however, the AGN selection wedges currently in use are heavily contaminated by star-forming galaxies, especially at high redshift. Using the large samples of luminous AGNs and high-redshift star-forming galaxies in COSMOS, we redefine the AGN selection criteria for use in deep IRAC surveys. The new IRAC criteria are designed to be both highly complete and reliable, and incorporate the best aspects of the current AGN selection wedges and of infrared power-law selection while excluding high-redshift star-forming galaxies selected via the BzK, distant red galaxy, Lyman-break galaxy, and submillimeter galaxy criteria. At QSO luminosities of log L2-10keV(erg s–1) ≥44, the new IRAC criteria recover 75% of the hard X-ray and IRAC-detected XMM-COSMOS sample, yet only 38% of the IRAC AGN candidates have X-ray counterparts, a fraction that rises to 52% in regions with Chandra exposures of 50-160 ks. X-ray stacking of the individually X-ray non-detected AGN candidates leads to a hard X-ray signal indicative of heavily obscured to mildly Compton-thick obscuration (log NH (cm–2) = 23.5 ± 0.4). While IRAC selection recovers a substantial fraction of luminous unobscured and obscured AGNs, it is incomplete to low-luminosity and host-dominated AGNs.

  16. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Itarte, Emilio, E-mail: emili.itarte@uab.es [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Meseguer, Anna, E-mail: ana.meseguer@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain)

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  17. Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas.

    Science.gov (United States)

    Wu, Chih-Ching; Hsu, Chia-Wei; Chen, Chi-De; Yu, Chia-Jung; Chang, Kai-Ping; Tai, Dar-In; Liu, Hao-Ping; Su, Wen-Hui; Chang, Yu-Sun; Yu, Jau-Song

    2010-06-01

    Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6-137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.

  18. A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells.

    Science.gov (United States)

    Wrighton, Kelly C; Agbo, Peter; Warnecke, Falk; Weber, Karrie A; Brodie, Eoin L; DeSantis, Todd Z; Hugenholtz, Philip; Andersen, Gary L; Coates, John D

    2008-11-01

    Significant effort is currently focused on microbial fuel cells (MFCs) as a source of renewable energy. Most studies concentrate on operation at mesophilic temperatures. However, anaerobic digestion studies have reported on the superiority of thermophilic operation and demonstrated a net energy gain in terms of methane yield. As such, our studies focused on MFC operation and microbiology at 55 degrees C. Over a 100-day operation, these MFCs were stable and achieved a power density of 37 mW m(-2) with a coulombic efficiency of 89%. To infer activity and taxonomic identity of dominant members of the electricity-producing community, we performed phylogenetic microarray and clone library analysis with small subunit ribosomal RNA (16S rRNA) and ribosomal RNA gene (16S rDNA). The results illustrated the dominance (80% of clone library sequences) of the Firmicutes in electricity production. Similarly, rRNA sequences from Firmicutes accounted for 50% of those taxa that increased in relative abundance from current-producing MFCs, implying their functional role in current production. We complemented these analyses by isolating the first organisms from a thermophilic MFC. One of the isolates, a Firmicutes Thermincola sp. strain JR, not only produced more current than known organisms (0.42 mA) in an H-cell system but also represented the first demonstration of direct anode reduction by a member of this phylum. Our research illustrates the importance of using a variety of molecular and culture-based methods to reliably characterize bacterial communities. Consequently, we revealed a previously unidentified functional role for Gram-positive bacteria in MFC current generation. PMID:18769460

  19. Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

    Science.gov (United States)

    Hjorth, Johannes J J; Dawitz, Julia; Kroon, Tim; Pires, Johny; Dassen, Valerie J; Berkhout, Janna A; Emperador Melero, Javier; Nadadhur, Aish G; Alevra, Mihai; Toonen, Ruud F; Heine, Vivi M; Mansvelder, Huibert D; Meredith, Rhiannon M

    2016-04-01

    Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers. PMID:26097169

  20. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna;

    2015-01-01

    85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted...... for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies.......Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...

  1. Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology

    Directory of Open Access Journals (Sweden)

    Marta Moreno

    2015-08-01

    Full Text Available Neural stem cells (NSCs reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state.

  2. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    OpenAIRE

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

  3. Remyelination of the Rat Spinal Cord by Transplantation of Identified Bone Marrow Stromal Cells

    OpenAIRE

    Akiyama, Yukinori; Radtke, Christine; Kocsis, Jeffery D.

    2002-01-01

    Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal cells were GFP expressing and immunopositive for collagen type I, fibronectin, and CD44. Tran...

  4. Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

    Institute of Scientific and Technical Information of China (English)

    Yu-Chieh Wang; Trevor R Leonardo; Ying Liu; Suzanne E Peterson; Louise C Laurent; Shinya Yamanaka; Jeanne F Loring; Masato Nakagawa; Ibon Garitaonandia; Ileana Slavin; Gulsah Altun; Robert M Lacharite; Kristopher L Nazor; Ha T Tran; Candace L Lynch

    2011-01-01

    Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications.Using lectin arrays,we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples,and identified a small group of iectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types.These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined,regardless of the laboratory of origin,the culture conditions,the somatic cell type reprogrammed,or the reprogramming method used.We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry,and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation.Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases,which may underlie these differences in protein glycosylation and lectin binding.Taken together,our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells,and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.

  5. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    NARCIS (Netherlands)

    S. James (Sally); J. Fox (James); F. Afsari (Farinaz); J. Lee (Jennifer); S. Clough (Sally); C. Knight (Charlotte); J. Ashmore (James); P. Ashton (Peter); O. Preham (Olivier); M.J. Hoogduijn (Martin); R.D.A.R. Ponzoni (Raquel De Almeida Rocha); Y. Hancock; M. Coles (Mark); P.G. Genever (Paul)

    2015-01-01

    textabstractBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis

  6. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

    NARCIS (Netherlands)

    Amps, Katherine; Andrews, Peter W.; Anyfantis, George; Armstrong, Lyle; Avery, Stuart; Baharvand, Hossein; Baker, Julie; Baker, Duncan; Munoz, Maria B.; Beil, Stephen; Benvenisty, Nissim; Ben-Yosef, Dalit; Biancotti, Juan-Carlos; Bosman, Alexis; Brena, Romulo Martin; Brison, Daniel; Caisander, Gunilla; Camarasa, Maria V.; Chen, Jieming; Chiao, Eric; Choi, Young Min; Choo, Andre B. H.; Collins, Daniel; Colman, Alan; Crook, Jeremy M.; Daley, George Q.; Dalton, Anne; De Sousa, Paul A.; Denning, Chris; Downie, Janet; Dvorak, Petr; Montgomery, Karen D.; Feki, Anis; Ford, Angela; Fox, Victoria; Fraga, Ana M.; Frumkin, Tzvia; Ge, Lin; Gokhale, Paul J.; Golan-Lev, Tamar; Gourabi, Hamid; Gropp, Michal; Lu Guangxiu, [No Value; Hampl, Ales; Harron, Katie; Healy, Lyn; Herath, Wishva; Holm, Frida; Hovatta, Outi; Hyllner, Johan; Inamdar, Maneesha S.; Irwanto, Astrid Kresentia; Ishii, Tetsuya; Jaconi, Marisa; Jin, Ying; Kimber, Susan; Kiselev, Sergey; Knowles, Barbara B.; Kopper, Oded; Kukharenko, Valeri; Kuliev, Anver; Lagarkova, Maria A.; Laird, Peter W.; Lako, Majlinda; Laslett, Andrew L.; Lavon, Neta; Lee, Dong Ryul; Lee, Jeoung Eun; Li, Chunliang; Lim, Linda S.; Ludwig, Tenneille E.; Ma, Yu; Maltby, Edna; Mateizel, Ileana; Mayshar, Yoav; Mileikovsky, Maria; Minger, Stephen L.; Miyazaki, Takamichi; Moon, Shin Yong; Moore, Harry; Mummery, Christine; Nagy, Andras; Nakatsuji, Norio; Narwani, Kavita; Oh, Steve K. W.; Oh, Sun Kyung; Olson, Cia; Otonkoski, Timo; Pan, Fei; Park, In-Hyun; Pells, Steve; Pera, Martin F.; Pereira, Lygia V.; Qi, Ouyang; Raj, Grace Selva; Reubinoff, Benjamin; Robins, Alan; Robson, Paul; Rossant, Janet; Salekdeh, Ghasem H.; Schulz, Thomas C.; Sermon, Karen; Mohamed, Jameelah Sheik; Shen, Hui; Sherrer, Eric; Sidhu, Kuldip; Sivarajah, Shirani; Skottman, Heli; Spits, Claudia; Stacey, Glyn N.; Strehl, Raimund; Strelchenko, Nick; Suemori, Hirofumi; Sun, Bowen; Suuronen, Riitta; Takahashi, Kazutoshi; Tuuri, Timo; Venu, Parvathy; Verlinsky, Yuri; Ward-van Oostwaard, Dorien; Weisenberger, Daniel J.; Wu, Yue; Yamanaka, Shinya; Young, Lorraine; Zhou, Qi

    2011-01-01

    The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphi

  7. Integrated transcriptomic and proteomic analysis identifies protein kinase CK2 as a key signaling node in an inflammatory cytokine network in ovarian cancer cells

    Science.gov (United States)

    Kulbe, Hagen; Iorio, Francesco; Chakravarty, Probir; Milagre, Carla S.; Moore, Robert; Thompson, Richard G.; Everitt, Gemma; Canosa, Monica; Montoya, Alexander; Drygin, Denis; Braicu, Ioana; Sehouli, Jalid; Saez-Rodriguez, Julio; Cutillas, Pedro R.; Balkwill, Frances R.

    2016-01-01

    We previously showed how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in ovarian cancer. This network, which we named the “TNF network”, has paracrine actions within the tumor microenvironment, influencing angiogenesis and the immune cell infiltrate. The aim of this study was to identify critical regulators in the signaling pathways of the TNF network in ovarian cancer cells that might be therapeutic targets. To achieve our aim, we used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis. Among the potential therapeutic kinase targets identified was the protein kinase Casein kinase II (CK2). Knockdown of CK2 expression in malignant cells by siRNA or treatment with the specific CK2 inhibitor CX-4945 significantly decreased Notch signaling and reduced constitutive cytokine release in ovarian cancer cell lines that expressed the TNF network as well as malignant cells isolated from high grade serous ovarian cancer ascites. The expression of the same cytokines was also inhibited after treatment with CX-4945 in a 3D organotypic model. CK2 inhibition was associated with concomitant inhibition of proliferative activity, reduced angiogenesis and experimental peritoneal ovarian tumor growth. In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer. PMID:26871292

  8. A Genetic Screen Identifies PITX1 as a Suppressor of RAS Activity and Tumorigenicity

    NARCIS (Netherlands)

    Kolfschoten, I.G.M.; Leeuwen, Bart van; Berns, K.; Mullenders, J.; Beijersbergen, R.L.; Bernards, R.A.; Voorhoeve, P.M.; Agami, R.

    2005-01-01

    Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or anothe

  9. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    Science.gov (United States)

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  10. A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.

    Science.gov (United States)

    Ao, Jie; Aldabbous, Mash'el; Notaro, Marysa J; Lojacono, Mark; Free, Stephen J

    2016-09-01

    A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation. PMID:27381444

  11. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    Science.gov (United States)

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  12. Multiple Lineages of Human Breast Cancer Stem/Progenitor Cells Identified by Profiling with Stem Cell Markers

    OpenAIRE

    Hwang-Verslues, Wendy W.; Wen-Hung Kuo; Po-Hao Chang; Chi-Chun Pan; Hsing-Hui Wang; Sheng-Ta Tsai; Yung-Ming Jeng; Jin-Yu Shew; Kung, John T.; Chung-Hsuan Chen; Lee, Eva Y-H. P.; King-Jen Chang; Wen-Hwa Lee

    2009-01-01

    Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vi...

  13. A Transcriptional Mechanism Integrating Inputs from Extracellular Signals to Activate Hippocampal Stem Cells

    OpenAIRE

    Andersen, Jimena; Urbán, Noelia; Achimastou, Angeliki; Ito, Ayako; Simic, Milesa; Ullom, Kristy; Martynoga, Ben; Lebel, Mélanie; Göritz, Christian; Frisén, Jonas; Nakafuku, Masato; Guillemot, François

    2014-01-01

    Summary The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate...

  14. An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain

    Science.gov (United States)

    Reinhart, Bonnie; Goins, William F; Harel, Asaff; Chaudhry, Suchita; Goss, James R; Yoshimura, Naoki; de Groat, William C; Cohen, Justus B; Glorioso, Joseph C

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1) is a pronociceptive cation channel involved in persistent inflammatory and neuropathic pain. Herpes simplex virus (HSV) vector expression of TRPV1 causes cell death in the presence of capsaicin, thereby completely blocking virus replication. Here we describe a selection system for negative regulators of TRPV1 based on rescue of virus replication. HSV-based coexpression of TRPV1 and a PC12 cell-derived cDNA library identified protein phosphatase 1α (PP1α) as a negative regulator of TRPV1, mimicking the activity of “poreless” (PL), a dominant-negative mutant of TRPV1. Vectors expressing PP1α or PL reduced thermal sensitivity following virus injection into rat footpads, but failed to reduce the nocifensive responses to menthol/icilin-activated cold pain or formalin, demonstrating that the activity identified in vitro is functional in vivo with a degree of specificity. This system should prove powerful for identifying other cellular factors that can inhibit ion channel activity. PMID:27382601

  15. A multi-omics approach identifies key hubs associated with cell type-specific responses of airway epithelial cells to staphylococcal alpha-toxin.

    Directory of Open Access Journals (Sweden)

    Erik Richter

    Full Text Available Responsiveness of cells to alpha-toxin (Hla from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects.

  16. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

    Directory of Open Access Journals (Sweden)

    James D Glover

    Full Text Available In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.

  17. CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Rouzbeh Taghizadeh

    Full Text Available A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

  18. Deletion of Scap in Alveolar Type II Cells Influences Lung Lipid Homeostasis and Identifies a Compensatory Role for Pulmonary Lipofibroblasts*S⃞

    OpenAIRE

    Besnard, Valérie; Wert, Susan E.; Stahlman, Mildred T.; Postle, Anthony D.; Xu, Yan; Ikegami, Machiko; Whitsett, Jeffrey A.

    2009-01-01

    Pulmonary function after birth is dependent upon surfactant lipids that reduce surface tension in the alveoli. The sterol-responsive element-binding proteins (SREBPs) are transcription factors regulating expression of genes controlling lipid homeostasis in many tissues. To identify the role of SREBPs in the lung, we conditionally deleted the SREBP cleavage-activating protein gene, Scap, in respiratory epithelial cells (ScapΔ/Δ) in vivo. Prior to birth (E18.5), dele...

  19. CD11b expression as a marker to distinguish between recently activated effector CD8(+) T cells and memory cells

    DEFF Research Database (Denmark)

    Christensen, Jeanette Erbo; Ørding Andreasen, Susanne; Christensen, Jan Pravsgaard;

    2001-01-01

    subset. Polyclonal virus-specific effector and memory CD8(+) T cells from lymphocytic choriomeningitis- and vesicular stomatitis virus-infected mice were visualized through staining for intracellular IFN-gamma or binding of MHC-peptide tetramers, and Mac-1 expression was evaluated. Naive T cells and most......CD8(+) T cells in different activation states have been difficult to identify phenotypically. In this study we have investigated whether Mac-1 (CD11b) expression can be used as a criterion to distinguish between recently activated effector cells and memory cells belonging to the CD8(+) T cell...... virus-specific memory CD8(+) T cells express little or no Mac-1 independent of the virus model employed. In contrast, the majority of CD8(+) T cells present during acute infection express a significant level of Mac-1 and, similarly, Mac-1 expression is found on secondary effectors generated in response...

  20. Identifying Key Features of Effective Active Learning: The Effects of Writing and Peer Discussion

    OpenAIRE

    Linton, Debra L.; Pangle, Wiline M.; Wyatt, Kevin H.; Powell, Karli N.; Sherwood, Rachel E.

    2014-01-01

    We investigated some of the key features of effective active learning by comparing the outcomes of three different methods of implementing active-learning exercises in a majors introductory biology course. Students completed activities in one of three treatments: discussion, writing, and discussion + writing. Treatments were rotated weekly between three sections taught by three different instructors in a full factorial design. The data set was analyzed by generalized linear mixed-effect model...

  1. Identifying Active Travel Behaviors in Challenging Environments Using GPS, Accelerometers, and Machine Learning Algorithms

    OpenAIRE

    Ellis, Katherine; Godbole, Suneeta; Marshall, Simon; Lanckriet, Gert; Staudenmayer, John; Kerr, Jacqueline

    2014-01-01

    Background: Active travel is an important area in physical activity research, but objective measurement of active travel is still difficult. Automated methods to measure travel behaviors will improve research in this area. In this paper, we present a supervised machine learning method for transportation mode prediction from global positioning system (GPS) and accelerometer data. Methods: We collected a dataset of about 150 h of GPS and accelerometer data from two research assistants follow...

  2. Identifying active travel behaviors in challenging environments using GPS, accelerometers and machine learning algorithms

    OpenAIRE

    Katherine eEllis; Suneeta eGodbole; Simon eMarshall; Gert eLanckriet; John eStaudenmayer; Jacqueline eKerr

    2014-01-01

    Background: Active travel is an important area in physical activity research, but objective measurement of active travel is still difficult. Automated methods to measure travel behaviors will improve research in this area. In this paper we present a supervised machine learning method for transportation mode prediction from GPS and accelerometer data. Methods: We collected a dataset of about 150 hours of GPS and accelerometer data from two research assistants following a protocol of prescribe...

  3. Microarray analysis of LTR retrotransposon silencing identifies Hdac1 as a regulator of retrotransposon expression in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Judith Reichmann

    Full Text Available Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.

  4. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    Directory of Open Access Journals (Sweden)

    Truernit Elisabeth

    2008-06-01

    Full Text Available Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP fluorescence was possible. Conclusion The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

  5. Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria.

    Science.gov (United States)

    Boyan, George; Posser, Susanne; Ludwig, Peter; Güntner, Michaela; Williams, Lez

    2004-04-01

    In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.

  6. Activation of cell divisions in legume nodulation

    DEFF Research Database (Denmark)

    Nadzieja, Marcin

    2016-01-01

    Leguminous plants engage into symbiotic relationships with soil bacteria, rhizobia, and develop root nodules. This process initiates with recognition of bacteria derived signalling molecules called nod factors. The subsequent events lead to symbiotic infection and, occurring in parallel, de novo...... was shown to require auxin signalling. Cytokinin, in contrast, exert a negative regulation of bacterial entry into the root. During organogenesis, auxin and cytokinin maxima are known to accompany nodule primordia development and together regulate progression through the cell cycle. Moreover, application...... observations of the DR5 reporter showed activity maxima situated in pericycle and endodermis cells specifically below infection sites. Using gravitropic bending auxin responses in the pericycle could be induced, specifically on the outer side of the gravitropic bend in uninoculated plants. When conducted...

  7. A simple way to identify non-viable cells within living plant tissue using confocal microscopy

    OpenAIRE

    Truernit Elisabeth; Haseloff Jim

    2008-01-01

    Abstract Background Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. Results Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy....

  8. Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers.

    Directory of Open Access Journals (Sweden)

    Wendy W Hwang-Verslues

    Full Text Available Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+/CD24(-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+/ESA(+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+/CD24(-/low, ESA(+, CD133(+, CXCR4(+ and PROCR(+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

  9. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  10. Selecting an optimal instrument to identify active ingredients of the motivational interviewing-process

    NARCIS (Netherlands)

    Dobber, Jos; Meijel, Berno van; Barkhof, Emile; Scholte op Reimer, Wilma; Latour, Corine; Peters, Ron; Linszen, Don

    2014-01-01

    Motivational Interviewing (MI) can effectively stimulate motivation for health behavior change, but the active ingredients of MI are not well known. To help clinicians further stimulate motivation, they need to know the active ingredients of MI. A psychometrically sound instrument is required to ide

  11. Identifying the Barriers and Facilitators to Participation in Physical Activity for Children with Down Syndrome

    Science.gov (United States)

    Barr, M.; Shields, N.

    2011-01-01

    Background: Many children with Down syndrome do not undertake the recommended amount of daily physical activity. The aim of this study was to explore the barriers and facilitators to physical activity for this group. Methods: Eighteen in-depth interviews were conducted with 20 parents (16 mothers, 4 fathers) of children with Down syndrome aged…

  12. Identifying types of physical activity with a single accelerometer: Evaluating laboratory trained algorithms in daily life

    NARCIS (Netherlands)

    Cuba Gyllensten, I.; Bonomi, A.G.

    2011-01-01

    Accurate identification of physical activity types has been achieved in laboratory conditions using single-site accelerometers and classification algorithms. This methodology is then applied to free-living subjects to determine activity behaviour. This study aimed at analysing the reproducibility of

  13. Polystyrene nanoparticles activate ion transport in human airway epithelial cells

    Directory of Open Access Journals (Sweden)

    McCarthy J

    2011-06-01

    Full Text Available J McCarthy1, X Gong2, D Nahirney2, M Duszyk2, MW Radomski11School of Pharmacy and Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Physiology, University of Alberta, Edmonton, Alberta, CanadaBackground: Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function.Methods: Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Cl- channels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches.Results: Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl- channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl- and HCO3- secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl- channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl- channels by the nanoparticles.Conclusion: This is the first study to identify

  14. A new marker set that identifies fetal cells in maternal circulation with high specificity

    DEFF Research Database (Denmark)

    Hatt, Lotte; Brinch, Marie; Singh, Ripudaman;

    2014-01-01

    OBJECTIVE: Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. METHOD: Blood samples from 78 normal pregnancies...

  15. Creation and validation of an instrument to identify nursing activities in pediatric wards: information for determining workload

    Directory of Open Access Journals (Sweden)

    Nanci Cristiano Santos

    2013-10-01

    Full Text Available The aim of this study was to develop and validate an instrument for identifying nursing activities performed in a pediatric ward and to provide a basis for defining the workload of these units. The instrument was developed by selecting the activities relevant to pediatric nursing from the Nursing Intervention Classification and then submitting them to a panel of judges for validation. The panel considered the selected activities relevant and representative of pediatric nursing practice. Now that representative activities for the nursing workload have been identified, we envision new studies to verify their usefulness in practice. Determining the amount of time each activity takes to perform will help to develop a system for measuring the workloads of nursing teams in pediatric wards.

  16. Snake venoms components with antitumor activity in murine melanoma cells

    International Nuclear Information System (INIS)

    Despite the constant advances in the treatment of cancer, this disease remains one of the main causes of mortality worldwide. So, the development of new treatment modalities is imperative. Snake venom causes a variety of biological effects because they constitute a complex mixture of substances as disintegrins, proteases (serine and metalo), phospholipases A2, L-amino acid oxidases and others. The goal of the present work is to evaluate a anti-tumor activity of some snake venoms fractions. There are several studies of components derived from snake venoms with this kind of activity. After fractionation of snake venoms of the families Viperidae and Elapidae, the fractions were assayed towards murine melanoma cell line B16-F10 and fibroblasts L929. The results showed that the fractions of venom of the snake Notechis ater niger had higher specificity and potential antitumor activity on B16-F10 cell line than the other studied venoms. Since the components of this venom are not explored yet coupled with the potential activity showed in this work, we decided to choose this venom to develop further studies. The cytotoxic fractions were evaluated to identify and characterize the components that showed antitumoral activity. Western blot assays and zymography suggests that these proteins do not belong to the class of metallo and serine proteinases. (author)

  17. Identifying A Molecular Phenotype for Bone Marrow Stromal Cells With In Vivo Bone Forming Capacity

    DEFF Research Database (Denmark)

    Larsen, Kenneth H; Frederiksen, Casper M; Burns, Jorge S;

    2009-01-01

    Abstract The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone forming capacity is not known. Thus, we employed DNA microarrays...... comparing two human bone marrow stromal cell (hBMSC) populations: one is capable of in vivo heterotopic bone formation (hBMSC-TERT(+Bone)) and the other is not (hBMSC-TERT(-Bone)). Compared to hBMSC-TERT(-Bone), the hBMSC-TERT(+Bone) cells had an increased over-representation of extracellular matrix genes...... (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT(-Bone) cells expressed a larger number of immune-response related genes (26% versus 8%). In order to test for the predictive...

  18. Critical narrative review to identify educational strategies promoting physical activity in preschool.

    Science.gov (United States)

    Kreichauf, S; Wildgruber, A; Krombholz, H; Gibson, E L; Vögele, C; Nixon, C A; Douthwaite, W; Moore, H J; Manios, Y; Summerbell, C D

    2012-03-01

    The aim of this narrative review is critically to evaluate educational strategies promoting physical activity that are used in the preschool setting in the context of obesity prevention programmes. Literature search was conducted between April and August 2010 in English and German databases (PubMED, PsychINFO, PSYNDEX, ERIC, FIS Bildung). Outcomes considered were time and intensity of physical activity, motor skills or measures of body composition. A total of 19 studies were included. Ten studies added physical activity lessons into their curriculum, one study provided more time for free play, eight studies focused on the social and play environment. Studies reporting positive outcomes implemented physical activity sessions that lasted at least 30 min d(-1). Several studies showed that children are most active in the first 10-15 min. The existence or installation of playground markings or fixed play equipment had no effect, whereas the presence or addition of portable play equipment was positively correlated with moderate-to-vigorous physical activity. Teacher training may be a key element for successful interventions. To overcome time constraints, a suggested solution is to integrate physical activity into daily routines and other areas of the preschool curriculum.

  19. OEFinder: a user interface to identify and visualize ordering effects in single-cell RNA-seq data

    OpenAIRE

    Leng, Ning; Choi, Jeea; Chu, Li-Fang; Thomson, James A.; Kendziorski, Christina; Stewart, Ron

    2016-01-01

    Summary: A recent article identified an artifact in multiple single-cell RNA-seq (scRNA-seq) datasets generated by the Fluidigm C1 platform. Specifically, Leng et al. showed significantly increased gene expression in cells captured from sites with small or large plate output IDs. We refer to this artifact as an ordering effect (OE). Including OE genes in downstream analyses could lead to biased results. To address this problem, we developed a statistical method and software called OEFinder to...

  20. Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells

    OpenAIRE

    Jaluria, Pratik; Betenbaugh, Michael; Konstantopoulos, Konstantinos; Frank, Bryan; Shiloach, Joseph

    2006-01-01

    The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical met...

  1. Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons

    NARCIS (Netherlands)

    M.P. Baggelaar; P.J.P. Chameau; V. Kantae; J. Hummel; K.L. Hsu; F. Janssen; T. van der Wel; M. Soethoudt; H. Deng; H. den Dulk; M. Allarà; B.I. Florea; V. Di Marzo; W.J. Wadman; C.G. Kruse; H.S. Overkleeft; T. Hankemeier; T.R. Werkman; B.F. Cravatt; M. van der Stelt

    2015-01-01

    Diacylglycerol lipase (DAGL)-alpha and -beta are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently l

  2. A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1.

    Directory of Open Access Journals (Sweden)

    Davide Danovi

    Full Text Available Glioblastoma multiforme (GBM is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS cells and genetically normal neural stem (NS cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101 as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1 (phospho T210, with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364 phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF(-/-, or p53(-/-, as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.

  3. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    Science.gov (United States)

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  4. Identifying active travel behaviors in challenging environments using GPS, accelerometers and machine learning algorithms

    Directory of Open Access Journals (Sweden)

    Katherine eEllis

    2014-04-01

    Full Text Available Background: Active travel is an important area in physical activity research, but objective measurement of active travel is still difficult. Automated methods to measure travel behaviors will improve research in this area. In this paper we present a supervised machine learning method for transportation mode prediction from GPS and accelerometer data. Methods: We collected a dataset of about 150 hours of GPS and accelerometer data from two research assistants following a protocol of prescribed trips consisting of five activities: bicycling, riding in a vehicle, walking, sitting, and standing. We extracted 49 features from 1-minute windows of this data. We compared the performance of several machine learning algorithms and chose a random forest algorithm to classify the transportation mode. We used a moving average output filter to smooth the output predictions over time. Results: The random forest algorithm achieved 89.8% cross-validated accuracy on this dataset. Adding the moving average filter to smooth output predictions increased the cross-validated accuracy to 91.9%. Conclusions: Machine learning methods are a viable approach for automating measurement of active travel, particularly for measuring travel activities that traditional accelerometer data processing methods misclassify, such as bicycling and vehicle travel.

  5. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  6. Elucidation of tonic and activated B-cell receptor signaling in Burkitt's lymphoma provides insights into regulation of cell survival.

    Science.gov (United States)

    Corso, Jasmin; Pan, Kuan-Ting; Walter, Roland; Doebele, Carmen; Mohr, Sebastian; Bohnenberger, Hanibal; Ströbel, Philipp; Lenz, Christof; Slabicki, Mikolaj; Hüllein, Jennifer; Comoglio, Federico; Rieger, Michael A; Zenz, Thorsten; Wienands, Jürgen; Engelke, Michael; Serve, Hubert; Urlaub, Henning; Oellerich, Thomas

    2016-05-17

    Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments. PMID:27155012

  7. Proton nuclear magnetic resonance spectroscopy unambiguously identifies different neural cell types.

    Science.gov (United States)

    Urenjak, J; Williams, S R; Gadian, D G; Noble, M

    1993-03-01

    Proton nuclear magnetic resonance (1H NMR) spectroscopy is a noninvasive technique that can provide information on a wide range of metabolites. Marked abnormalities of 1H NMR brain spectra have been reported in patients with neurological disorders, but their neurochemical implications may be difficult to appreciate because NMR data are obtained from heterogeneous tissue regions composed of several cell populations. The purpose of this study was to examine the 1H NMR profile of major neural cell types. This information may be helpful in understanding the metabolic abnormalities detected by 1H NMR spectroscopy. Extracts of cultured cerebellar granule neurons, cortical astrocytes, oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, oligodendrocytes, and meningeal cells were analyzed. The purity of the cultured cells was > 95% with all the cell lineages, except for neurons (approximately 90%). Although several constituents (creatine, choline-containing compounds, lactate, acetate, succinate, alanine, glutamate) were ubiquitously detectable with 1H NMR, each cell type had distinctive qualitative and/or quantitative features. Our most unexpected finding was a large amount of N-acetyl-aspartate (NAA) in O-2A progenitors. This compound, consistently detected by 1H NMR in vivo, was previously thought to ne present only in neurons. The finding that meningeal cells have an alanine:creatine ratio three to four times higher than astrocytes, neurons, or oligodendrocytes is in agreement with observations that meningiomas express a higher alanine:creatine ratio than gliomas. The data suggest that each individual cell type has a characteristic metabolic pattern that can be discriminated by 1H NMR, even by looking at only a few metabolites (e.g., NAA, glycine, beta-hydroxybutyrate).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8441018

  8. Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

    Science.gov (United States)

    Haage, Amanda; Schneider, Ian C

    2014-08-01

    The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

  9. The complexities of identifying a cell of origin for human prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Gail P Risbridger; Renea A Taylor

    2011-01-01

    @@ Prostate cancer(PCa)is the second most common malignancy in men1 and ifloca-lized or confined to the gland at diagnosis,the choice of treatment includes surgery,radiation or watchful waiting.If and when the disease spreads,androgen blockade is effective but inevitably relapse occurs,resulting in incurable castrate-resistant PCa.Understanding the etiology of PCa will underpin the development of better treatment options In particular,the identity of the cell type(s)that are the origin of PCa(or cancer-initiating cells)will enable them to become therapeutic targets that could lead to newer and more sophisticated treatment options.These cells of origin are defined as epithelial cells in the normal prostate,susceptible to malignant transformation and therefore capable of initiating tumourigenesis.However,their identity is unknown.

  10. Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures

    DEFF Research Database (Denmark)

    Junker, Niels; Kvistborg, Pia; Køllgaard, Tania;

    2012-01-01

    Ex vivo expanded tumor infiltrating lymphocytes (TILs) from malignant melanoma (MM) and head & neck squamous cell carcinoma (HNSCC) share a similar oligoclonal composition of T effector memory cells, with HLA class I restricted lysis of tumor cell lines. In this study we show that ex vivo expanded...... TILs from MM and HNSCC demonstrate a heterogeneous composition in frequency and magnitude of tumor associated antigen specific populations by Elispot IFN¿ quantitation. TILs from MM and HNSCC shared reactivity towards NY ESO-1, cyclin B1 and Bcl-x derived peptides. Additionally we show that dominating...... the heterogeneous tumors upon adoptive transfer; increasing the probability of tumor control by minimizing immune evasion by tumor cell escape variants....

  11. Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette;

    2002-01-01

    The purpose of this study was to characterize the effects of human retinal pigment epithelial (RPE) cells on activated T cells. Activated T cells were cocultured with adult and foetal human RPE cells whereafter apoptosis and proliferation were determined by flow cytometry and (3)H......-Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact...

  12. Identifying active functionalities on few-layered graphene catalysts for oxidative dehydrogenation of isobutane.

    Science.gov (United States)

    Dathar, Gopi Krishna Phani; Tsai, Yu-Tung; Gierszal, Kamil; Xu, Ye; Liang, Chengdu; Rondinone, Adam J; Overbury, Steven H; Schwartz, Viviane

    2014-02-01

    The general consensus in the studies of nanostructured carbon catalysts for oxidative dehydrogenation (ODH) of alkanes to olefins is that the oxygen functionalities generated during synthesis and reaction are responsible for the catalytic activity of these nanostructured carbons. Identification of the highly active oxygen functionalities would enable engineering of nanocarbons for ODH of alkanes. Few-layered graphenes were used as model catalysts in experiments to synthesize reduced graphene oxide samples with varying oxygen concentrations, to characterize oxygen functionalities, and to measure the activation energies for ODH of isobutane. Periodic density functional theory calculations were performed on graphene nanoribbon models with a variety of oxygen functionalities at the edges to calculate their thermal stability and to model reaction mechanisms for ODH of isobutane. Comparing measured and calculated thermal stability and activation energies leads to the conclusion that dicarbonyls at the zigzag edges and quinones at armchair edges are appropriately balanced for high activity, relative to other model functionalities considered herein. In the ODH of isobutane, both dehydrogenation and regeneration of catalytic sites are relevant at the dicarbonyls, whereas regeneration is facile compared with dehydrogenation at quinones. The catalytic mechanism involves weakly adsorbed isobutane reducing functional oxygen and leaving as isobutene, and O2 in the feed, weakly adsorbed on the hydrogenated functionality, reacting with that hydrogen and regenerating the catalytic sites.

  13. Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Morris, Sydney E; Moody, Sally A; Nemes, Peter

    2016-06-21

    Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal-vegetal and dorsal-ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left-right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate-triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p < 0.05 and fold change ≥1.5). These metabolites were enriched in the arginine-proline metabolic pathway in the right, but not the left D1 cells. Besides providing analytical benefits for single-cell HRMS, this work provides new metabolic data on the establishment of normal body asymmetry in the early developing embryo. PMID:27004603

  14. 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.

  15. Natural killer cell immunomodulation: targeting activating, inhibitory, and co-stimulatory receptor signaling for cancer immunotherapy

    Directory of Open Access Journals (Sweden)

    Cariad eChester

    2015-12-01

    Full Text Available There is compelling clinical and experimental evidence to suggest natural killer (NK cells play a critical role in the recognition and eradication of tumors. Efforts at using NK cells as antitumor agents began over two decades ago, but recent advances in elucidating NK cell biology have accelerated the development of NK cell-targeting therapeutics. NK cell activation and the triggering of effector functions is governed by a complex set of activating and inhibitory receptors. In the early phases of cancer immune surveillance, NK cells directly identify and lyse cancer cells. Nascent transformed cells elicit NK cell activation and are eliminated. However, as tumors progress, cancerous cells develop immunosuppressive mechanisms that circumvent NK cell-mediated killing, allowing for tumor escape and proliferation. Therapeutic intervention aims to reverse tumor-induced NK cell suppression and sustain NK cells’ tumorlytic capacities. Here, we review tumor-NK cell interactions, discuss the mechanisms by which NK cells generate an antitumor immune response, and discuss NK cell-based therapeutic strategies targeting activating, inhibitory, and costimulatory receptors.

  16. Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

    Directory of Open Access Journals (Sweden)

    Anantha Koteswararao Kanugula

    Full Text Available Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.

  17. Fas Activation in Alveolar Epithelial Cells Induces KC (CXCL1) Release by a MyD88-Dependent Mechanism

    OpenAIRE

    Farnand, Alex W.; Eastman, Alison J.; Herrero, Raquel; Hanson, Josiah F.; Mongovin, Steve; Altemeier, William A.; Matute-Bello, Gustavo

    2011-01-01

    Activation of the Fas/Fas ligand (FasL) system is associated with activation of apoptotic and proinflammatory pathways that lead to the development of acute lung injury. Previous studies in chimeric mice and macrophage-depleted mice suggested that the main effector cell in Fas-mediated lung injury is not a myeloid cell, but likely an epithelial cell. The goal of this study was to determine whether epithelial cells release proinflammatory cytokines after Fas activation, and to identify the rel...

  18. Response Properties of a Newly Identified Tristratified Narrow Field Amacrine Cell in the Mouse Retina.

    Directory of Open Access Journals (Sweden)

    G S Newkirk

    Full Text Available Amacrine cells were targeted for whole cell recording using two-photon fluorescence microscopy in a transgenic mouse line in which the promoter for dopamine receptor 2 drove expression of green fluorescent protein in a narrow field tristratified amacrine cell (TNAC that had not been studied previously. Light evoked a multiphasic response that was the sum of hyperpolarizing and depolarization synaptic inputs consistent with distinct dendritic ramifications in the off and on sublamina of the inner plexiform layer. The amplitude and waveform of the response, which consisted of an initial brief hyperpolarization at light onset followed by recovery to a plateau potential close to dark resting potential and a hyperpolarizing response at the light offset varied little over an intensity range from 0.4 to ~10^6 Rh*/rod/s. This suggests that the cell functions as a differentiator that generates an output signal (a transient reduction in inhibitory input to downstream retina neurons that is proportional to the derivative of light input independent of its intensity. The underlying circuitry appears to consist of rod and cone driven on and off bipolar cells that provide direct excitatory input to the cell as well as to GABAergic amacrine cells that are synaptically coupled to TNAC. Canonical reagents that blocked excitatory (glutamatergic and inhibitory (GABA and glycine synaptic transmission had effects on responses to scotopic stimuli consistent with the rod driven component of the proposed circuit. However, responses evoked by photopic stimuli were paradoxical and could not be interpreted on the basis of conventional thinking about the neuropharmacology of synaptic interactions in the retina.

  19. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

    Directory of Open Access Journals (Sweden)

    József Jászai

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  20. Identifying subgroups among poor prognosis patients with nonseminomatous germ cell cancer by tree modelling: a validation study.

    NARCIS (Netherlands)

    M.R. van Dijk (Merel); E.W. Steyerberg (Ewout); S.P. Stenning; J.D.F. Habbema (Dik)

    2004-01-01

    textabstractBACKGROUND: In order to target intensive treatment strategies for poor prognosis patients with non-seminomatous germ cell cancer, those with the poorest prognosis should be identified. These patients might profit most from more intensive treatment strategies. For this p

  1. Comparative In Vitro Activities of Ertapenem (MK-0826) against 469 Less Frequently Identified Anaerobes Isolated from Human Infections

    OpenAIRE

    Goldstein, Ellie J. C.; Citron, Diane M.; Merriam, C. Vreni; Warren, Yumi A.; Tyrrell, Kerin L.; Fernandez, Helen

    2002-01-01

    We studied the in vitro activity of ertapenem against 469 less frequently identified anaerobes from 11 genera and 52 species isolated from human infections. Ertapenem was uniformly active against 460 of 469 (98%) strains at concentrations of ≤4 μg/ml. Only 4 of 14 Clostridium difficile, 1 of 11 Clostridium innocuum, and 4 of 6 Lactobacillus sp. strains required ertapenem concentrations of ≥8 μg/ml for inhibition.

  2. Using Concept Mapping to Identify Action Steps for Physical Activity Promotion in Cancer Treatment

    Science.gov (United States)

    Fitzpatrick, Sean Joseph; Zizzi, Sam J.

    2014-01-01

    Background: The benefits of exercise during and after cancer treatment represent research areas that have received increased attention throughout the past 2 decades. Numerous benefits have been observed for cancer survivors who are physically active, yet oncologists have been slow to incorporate exercise counseling into practice. Purpose: The…

  3. 3D Printing in Instructional Settings: Identifying a Curricular Hierarchy of Activities

    Science.gov (United States)

    Brown, Abbie

    2015-01-01

    A report of a year-long study in which the author engaged in 3D printing activity in order to determine how to facilitate and support skill building, concept attainment, and increased confidence with its use among teachers. Use of 3D printing tools and their applications in instructional settings are discussed. A hierarchy of 3D printing…

  4. A High-Throughput Screen Identifies a New Natural Product with Broad-Spectrum Antibacterial Activity

    OpenAIRE

    Patrick Ymele-Leki; Shugeng Cao; Jared Sharp; Kathleen G Lambert; McAdam, Alexander J.; Husson, Robert N.; Giselle Tamayo; Jon Clardy; Watnick, Paula I.

    2012-01-01

    Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms t...

  5. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...... and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated....

  6. Alpha-fetoprotein, identified as a novel marker for the antioxidant effect of placental extract, exhibits synergistic antioxidant activity in the presence of estradiol.

    Directory of Open Access Journals (Sweden)

    Hye Yeon Choi

    Full Text Available Placenta, as a reservoir of nutrients, has been widely used in medical and cosmetic materials. Here, we focused on the antioxidant properties of placental extract and attempted to isolate and identify the main antioxidant factors. Porcine placental extracts were prepared through homogenization or acid hydrolysis, and their antioxidant activity was investigated in the human keratinocyte HaCaT cell line. Treatment with homogenized placental extract (H-PE increased the cell viability of H2O2-treated HaCaT cells more than two-fold. H-PE treatment suppressed H2O2-induced apoptotic and necrotic cell death and decreased intracellular ROS levels in H2O2-treated HaCaT cells. The antioxidant factors in H-PE were found to be thermo-unstable and were thus expected to include proteins. The candidate antioxidant proteins were fractionated with cation-exchange, anion-exchange, and size-exclusion chromatography, and the antioxidant properties of the chromatographic fractions were investigated. We obtained specific antioxidant fractions that suppressed ROS generation and ROS-induced DNA strand breaks. From silver staining and MALDI-TOF analyses, alpha-fetoprotein (AFP precursor was identified as a main marker for the antioxidant effect of H-PE. Purified AFP or ectopically expressed AFP exhibited synergistic antioxidant activity in the presence of estradiol. Taken together, our data suggest that AFP, a serum glycoprotein produced at high levels during fetal development, is a novel marker protein for the antioxidant effect of the placenta that exhibits synergistic antioxidant activity in the presence of estradiol.

  7. A newly identified essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast.

    Directory of Open Access Journals (Sweden)

    Laurence Vernis

    Full Text Available A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H(2O(2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

  8. Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

    Institute of Scientific and Technical Information of China (English)

    Xing'an Li; Yinjiu Zhang; Yihong Hu; Ming Chang; Tao Liu; Danping Wang; Yu Zhang; Lei Zhang; Linsen Hu

    2008-01-01

    Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled.Protcomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and caldum-binding protein I were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.

  9. 4D Tumorigenesis Model for Quantitating Coalescence, Directed Cell Motility and Chemotaxis, Identifying Unique Cell Behaviors, and Testing Anticancer Drugs.

    Science.gov (United States)

    Kuhl, Spencer; Voss, Edward; Scherer, Amanda; Lusche, Daniel F; Wessels, Deborah; Soll, David R

    2016-01-01

    A 4D high-resolution computer-assisted reconstruction and motion analysis system has been developed and applied to the long-term (14-30 days) analysis of cancer cells migrating and aggregating within a 3D matrix. 4D tumorigenesis models more closely approximate the tumor microenvironment than 2D substrates and, therefore, are improved tools for elucidating the interactions within the tumor microenvironment that promote growth and metastasis. The model we describe here can be used to analyze the growth of tumor cells, aggregate coalescence, directed cell motility and chemotaxis, matrix degradation, the effects of anticancer drugs, and the behavior of immune and endothelial cells mixed with cancer cells. The information given in this chapter is also intended to acquaint the reader with computer-assisted methods and algorithms that can be used for high-resolution 3D reconstruction and quantitative motion analysis. PMID:27271907

  10. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    DEFF Research Database (Denmark)

    Ali; Hamam; Alfayez;

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including...... abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell...... or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating...

  11. Proteomics-identified Bvg-activated autotransporters protect against bordetella pertussis in a mouse model.

    Science.gov (United States)

    de Gouw, Daan; Gouw, Daan de; de Jonge, Marien I; Jonge, Marien I de; Hermans, Peter W M; Wessels, Hans J C T; Zomer, Aldert; Berends, Alinda; Pratt, Catherine; Berbers, Guy A; Mooi, Frits R; Diavatopoulos, Dimitri A

    2014-01-01

    Pertussis is a highly infectious respiratory disease of humans caused by the bacterium Bordetella pertussis. Despite high vaccination coverage, pertussis has re-emerged globally. Causes for the re-emergence of pertussis include limited duration of protection conferred by acellular pertussis vaccines (aP) and pathogen adaptation. Pathogen adaptations involve antigenic divergence with vaccine strains, the emergence of strains which show enhanced in vitro expression of a number of virulence-associated genes and of strains that do not express pertactin, an important aP component. Clearly, the identification of more effective B. pertussis vaccine antigens is of utmost importance. To identify novel antigens, we used proteomics to identify B. pertussis proteins regulated by the master virulence regulatory system BvgAS in vitro. Five candidates proteins were selected and it was confirmed that they were also expressed in the lungs of naïve mice seven days after infection. The five proteins were expressed in recombinant form, adjuvanted with alum and used to immunize mice as stand-alone antigens. Subsequent respiratory challenge showed that immunization with the autotransporters Vag8 and SphB1 significantly reduced bacterial load in the lungs. Whilst these antigens induced strong opsonizing antibody responses, we found that none of the tested alum-adjuvanted vaccines - including a three-component aP - reduced bacterial load in the nasopharynx, suggesting that alternative immunological responses may be required for efficient bacterial clearance from the nasopharynx.

  12. Novel Morphologic and Genetic Analysis of Cancer Cells in a 3D Microenvironment Identifies STAT3 as a Regulator of Tumor Permeability Barrier Function.

    Science.gov (United States)

    Park, Min Chul; Jeong, Hyobin; Son, Sung Hwa; Kim, YounHa; Han, Daeyoung; Goughnour, Peter C; Kang, Taehee; Kwon, Nam Hoon; Moon, Hyo Eun; Paek, Sun Ha; Hwang, Daehee; Seol, Ho Jun; Nam, Do-Hyun; Kim, Sunghoon

    2016-03-01

    Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

  13. Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas

    OpenAIRE

    Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-sound; Ailles, Laurie; Moghal, Nadeem

    2014-01-01

    Background The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers l...

  14. Anti-inflammatory activity of mushrooms extracts, identified phenolic acids and their possible metabolites

    OpenAIRE

    Taofiq, Oludemi; Calhelha, Ricardo C.; Heleno, Sandrina A.; Barros, Lillian; Martins, Anabela; Abreu, Rui M.V.; Queiroz, Maria João R. P.; Isabel C. F. R. Ferreira

    2015-01-01

    Mushrooms are rich sources of many bioactive compounds, such as phenolic acids, that play an important role in the organism, acting as antioxidants, antitumors, antimicrobials, immunomodulators, among others. However, their anti-inflammatory activity has not been deeply studied. In the present study, the ethanolic extracts of fourteen edible mushroom species were firstly characterized in terms of phenolic acids and related compounds by HPLC-PDA, followed by the study of the ant...

  15. Natural killer cell activity during premedication, anaesthesia and surgery

    DEFF Research Database (Denmark)

    Tønnesen, E; Mickley, H; Grunnet, N

    1983-01-01

    Natural killer (NK) cell activity of peripheral blood mononuclear cells was measured against K-562 target cells in a 51Cr release assay in eight patients undergoing total hip replacement surgery. Eight consecutive blood samples were taken from each patient. A significant increase of NK cell...... days. The findings of this study indicate that premedication, anaesthesia and surgery cause a rapid and transient increase in NK cell activity, followed by a decline in activity postoperatively. The transient increase in activity may be explained by mobilization of natural killer cells from extravasal...... activity was observed after premedication with diazepam per os. The activity increased further during a combined anaesthesia (thiopentone + N2O + O2 + buprenorphene + pancuronium) and remained increased during surgery. Postoperatively, NK cell activity fell and remained depressed for a period of at least 5...

  16. Relationship between somatostatin receptors and activation of hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳

    2004-01-01

    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  17. Identifying and Enhancing the Strengths of Gifted Learners, K-8: Easy-to-Use Activities and Lessons

    Science.gov (United States)

    Maccagnano, Ann Marie

    2007-01-01

    Educators can identify children's strengths early on and gain insight into each student's unique abilities by using the numerous ideas and informal assessments in this exciting guide. Gifted and talented specialist Ann Maccagnano offers K-8 teachers challenging activities and engaging lessons to develop and nurture gifted learners' talents.…

  18. The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes.

    Science.gov (United States)

    Hube, Florent; Myal, Yvonne; Leygue, Etienne

    2006-05-01

    Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.

  19. Pancreatic and pulmonary mast cells activation during experimental acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Inmaculada; Lopez-Font; Sabrina; Gea-Sorlí; Enrique; de-Madaria; Luis; M; Gutiérrez; Miguel; Pérez-Mateo; Daniel; Closa

    2010-01-01

    AIM:To study the activation of pancreatic and pulmonary mast cells and the effect of mast cell inhibition on the activation of peritoneal and alveolar macrophages during acute pancreatitis.METHODS:Pancreatitis was induced by intraductal infusion of 5% sodium taurodeoxycholate in rats.The mast cell inhibitor cromolyn was administered intraperitoneally(i.p.) 30 min before pancreatitis induction.The pancreatic and pulmonary tissue damage was evaluated histologically and mast cells and their state of activation...

  20. A Complementary Bioinformatics Approach to Identify Potential Plant Cell Wall Glycosyltransferase-Encoding Genes

    DEFF Research Database (Denmark)

    Egelund, Jack; Skjøt, Michael; Geshi, Naomi;

    2004-01-01

    . Although much is known with regard to composition and fine structures of the plant CW, only a handful of CW biosynthetic GT genes-all classified in the CAZy system-have been characterized. In an effort to identify CW GTs that have not yet been classified in the CAZy database, a simple bioinformatics...

  1. CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis.

    Science.gov (United States)

    Yoshida, Noriaki; Miyoshi, Hiroaki; Kato, Takeharu; Sakata-Yanagimoto, Mamiko; Niino, Daisuke; Taniguchi, Hiroaki; Moriuchi, Yukiyoshi; Miyahara, Masaharu; Kurita, Daisuke; Sasaki, Yuya; Shimono, Joji; Kawamoto, Keisuke; Utsunomiya, Atae; Imaizumi, Yoshitaka; Seto, Masao; Ohshima, Koichi

    2016-04-01

    Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology.

  2. Mechanism of Drug Resistance Identified in Human Lung Adenocarcinoma Cell Line SPC-A1 Selected for Resistance to Docetaxel

    Institute of Scientific and Technical Information of China (English)

    Hai Sun; Long-bang Chen

    2009-01-01

    Objective: To investigate the mechanism of resistance to docetaxel in human lung cancer.Methods: Human lung carcinoma SPC-A1/Docetaxel cells were derived from parental SPC-A1 cells by continuous exposure to increasing concentration of docetaxel. The drug sensitivity was measured by MTT assay in vitro. The cDNA microarray identified a set of differentially expressed genes, and some genes were confirmed by RT-PCR. P-glycoprotein level was measured by flow cytometry analysis.Results: The results of drug sensitivity measured by MTT assay showed that SPC-A1/Docetaxel cells were 13.2-fold resistant to docetaxel and cross-resistant at varying levels to other drugs. The cDNA microarray results identified a set of differentially expressed genes, which showed 428 genes that were up-regulated and 506 genes that were down-regulated in SPC-A1/Docetaxel cells, and some genes were confirmed by RT-PCR. Flow cytometry analysis suggests expression of P-glycoprotein (P-gp) was more abundant in SPC-A1/Docetaxel cells than in the parental cells and docetaxel selection reduces the apoptotic response.Conclusion: The results suggest that docetaxel selection led to changes in gene expression that contribute to the multidrug resistance phenotype.

  3. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  4. MicroRNA Expression Profiling Identifies Molecular Diagnostic Signatures for Anaplastic Large Cell Lymphoma

    DEFF Research Database (Denmark)

    Liu, Cuiling; Iqbal, Javeed; Teruya-Feldstein, Julie;

    2013-01-01

    Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimm...

  5. Does Fetal antigen 1 (FA1) identify cells with regenerative, endocrine and neuroendocrine potentials?

    DEFF Research Database (Denmark)

    Jensen, Charlotte Floridon; Jensen, Charlotte Harken; Thorsen, Poul;

    2000-01-01

    in the subcellular localisation indicating differential post-translational/post-transcriptional modifications during fetal development. FA1 may be a new marker of cellular subtypes with a regenerative potential and of specific cells with endocrine or neuroendocrine functions. Udgivelsesdato: 2000-Aug...

  6. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

    NARCIS (Netherlands)

    Peifer, Martin; Fernandez-Cuesta, Lynnette; Sos, Martin L.; George, Julie; Seidel, Danila; Kasper, Lawryn H.; Plenker, Dennis; Leenders, Frauke; Sun, Ruping; Zander, Thomas; Menon, Roopika; Koker, Mirjam; Dahmen, Ilona; Mueller, Christian; Di Cerbo, Vincenzo; Schildhaus, Hans-Ulrich; Altmueller, Janine; Baessmann, Ingelore; Becker, Christian; de Wilde, Bram; Vandesompele, Jo; Boehm, Diana; Ansen, Sascha; Gabler, Franziska; Wilkening, Ines; Heynck, Stefanie; Heuckmann, Johannes M.; Lu, Xin; Carter, Scott L.; Cibulskis, Kristian; Banerji, Shantanu; Getz, Gad; Park, Kwon-Sik; Rauh, Daniel; Gruetter, Christian; Fischer, Matthias; Pasqualucci, Laura; Wright, Gavin; Wainer, Zoe; Russell, Prudence; Petersen, Iver; Chen, Yuan; Stoelben, Erich; Ludwig, Corinna; Schnabel, Philipp; Hoffmann, Hans; Muley, Thomas; Brockmann, Michael; Engel-Riedel, Walburga; Muscarella, Lucia A.; Fazio, Vito M.; Groen, Harry; Timens, Wim; Sietsma, Hannie; Thunnissen, Erik; Smit, Egbert; Heideman, Danielle A. M.; Snijders, Peter J. F.; Cappuzzo, Federico; Ligorio, Claudia; Damiani, Stefania; Field, John; Solberg, Steinar; Brustugun, Odd Terje; Lund-Iversen, Marius; Saenger, Joerg; Clement, Joachim H.; Soltermann, Alex; Moch, Holger; Weder, Walter; Solomon, Benjamin; Soria, Jean-Charles; Validire, Pierre; Besse, Benjamin; Brambilla, Elisabeth; Brambilla, Christian; Lantuejoul, Sylvie; Lorimier, Philippe; Schneider, Peter M.; Hallek, Michael; Pao, William; Meyerson, Matthew; Sage, Julien; Shendure, Jay; Schneider, Robert; Buettner, Reinhard; Wolf, Juergen; Nuernberg, Peter; Perner, Sven; Heukamp, Lukas C.; Brindle, Paul K.; Haas, Stefan; Thomas, Roman K.

    2012-01-01

    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis(1-3). We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 +/- 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analys

  7. Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

    Directory of Open Access Journals (Sweden)

    Bianca Schmid

    2015-12-01

    Full Text Available Dengue virus (DENV is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

  8. Identifying the rules of engagement enabling leukocyte rolling, activation, and adhesion.

    Directory of Open Access Journals (Sweden)

    Jonathan Tang

    2010-02-01

    Full Text Available The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte-surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte-surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1 but not LFA1 rebinding (to ICAM1 was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and

  9. Identifying the rules of engagement enabling leukocyte rolling, activation, and adhesion.

    Science.gov (United States)

    Tang, Jonathan; Hunt, C Anthony

    2010-02-19

    The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte-surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte-surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1) but not LFA1 rebinding (to ICAM1) was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and ICAM1 object

  10. Models of self-peptide sampling by developing T cells identify candidate mechanisms of thymic selection.

    Directory of Open Access Journals (Sweden)

    Iren Bains

    Full Text Available Conventional and regulatory T cells develop in the thymus where they are exposed to samples of self-peptide MHC (pMHC ligands. This probabilistic process selects for cells within a range of responsiveness that allows the detection of foreign antigen without excessive responses to self. Regulatory T cells are thought to lie at the higher end of the spectrum of acceptable self-reactivity and play a crucial role in the control of autoimmunity and tolerance to innocuous antigens. While many studies have elucidated key elements influencing lineage commitment, we still lack a full understanding of how thymocytes integrate signals obtained by sampling self-peptides to make fate decisions. To address this problem, we apply stochastic models of signal integration by T cells to data from a study quantifying the development of the two lineages using controllable levels of agonist peptide in the thymus. We find two models are able to explain the observations; one in which T cells continually re-assess fate decisions on the basis of multiple summed proximal signals from TCR-pMHC interactions; and another in which TCR sensitivity is modulated over time, such that contact with the same pMHC ligand may lead to divergent outcomes at different stages of development. Neither model requires that T(conv and T(reg are differentially susceptible to deletion or that the two lineages need qualitatively different signals for development, as have been proposed. We find additional support for the variable-sensitivity model, which is able to explain apparently paradoxical observations regarding the effect of partial and strong agonists on T(conv and T(reg development.

  11. Computational modeling of glucose transport in pancreatic β-cells identifies metabolic thresholds and therapeutic targets in diabetes.

    Directory of Open Access Journals (Sweden)

    Camilla Luni

    Full Text Available Pancreatic β-cell dysfunction is a diagnostic criterion of Type 2 diabetes and includes defects in glucose transport and insulin secretion. In healthy individuals, β-cells maintain plasma glucose concentrations within a narrow range in concert with insulin action among multiple tissues. Postprandial elevations in blood glucose facilitate glucose uptake into β-cells by diffusion through glucose transporters residing at the plasma membrane. Glucose transport is essential for glycolysis and glucose-stimulated insulin secretion. In human Type 2 diabetes and in the mouse model of obesity-associated diabetes, a marked deficiency of β-cell glucose transporters and glucose uptake occurs with the loss of glucose-stimulated insulin secretion. Recent studies have shown that the preservation of glucose transport in β-cells maintains normal insulin secretion and blocks the development of obesity-associated diabetes. To further elucidate the underlying mechanisms, we have constructed a computational model of human β-cell glucose transport in health and in Type 2 diabetes, and present a systems analysis based on experimental results from human and animal studies. Our findings identify a metabolic threshold or "tipping point" whereby diminished glucose transport across the plasma membrane of β-cells limits intracellular glucose-6-phosphate production by glucokinase. This metabolic threshold is crossed in Type 2 diabetes and results in β-cell dysfunction including the loss of glucose stimulated insulin secretion. Our model further discriminates among molecular control points in this pathway wherein maximal therapeutic intervention is achieved.

  12. Hyperoxia Inhibits T Cell Activation in Mice

    Science.gov (United States)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  13. Enhancement of the non-invasive electroenterogram to identify intestinal pacemaker activity

    International Nuclear Information System (INIS)

    Surface recording of electroenterogram (EEnG) is a non-invasive method for monitoring intestinal myoelectrical activity. However, surface EEnG is seriously affected by a variety of interferences: cardiac activity, respiration, very low frequency components and movement artefacts. The aim of this study is to eliminate respiratory interference and very low frequency components from external EEnG recording by means of empirical mode decomposition (EMD), so as to obtain more robust indicators of intestinal pacemaker activity from the external EEnG signal. For this purpose, 11 recording sessions were performed in an animal model under fasting conditions and in each individual session the myoelectrical signal was recorded simultaneously in the intestinal serosa and the external abdominal surface in physiological states. Various parameters have been proposed for evaluating the efficacy of the method in reducing interferences: the signal-to-interference ratio (S/I ratio), attenuation of the target and interference signals, the normal slow wave percentage and the stability of the dominant frequency (DF) of the signal. The results show that the S/I ratio of the processed signals is significantly greater than the original values (9.66 ± 4.44 dB versus 1.23 ± 5.13 dB), while the target signal was barely attenuated (−0.63 ± 1.02 dB). The application of the EMD method also increased the percentage of the normal slow wave to 100% in each individual session and enabled the stability of the DF of the external signal to be increased considerably. Furthermore, the variation coefficient of the DF derived from the external processed signals is comparable to the coefficient obtained using internal recordings. Therefore, the EMD method could be a very useful tool to improve the quality of external EEnG recording in the low frequency range and therefore to obtain more robust indicators of the intestinal pacemaker activity from non-invasive EEnG recordings

  14. Distinct developmental defense activations in barley embryos identified by transcriptome profiling

    DEFF Research Database (Denmark)

    Nielsen, ME; Lok, F; Nielsen, Henrik Bjørn

    2006-01-01

    analyses of > 22,000 genes, which together with measurements of jasmonic acid and salicylic acid during embryo development provide new information on the initiation in the developing barley embryo of at least two distinct types of developmental defense activation (DDA). Early DDA is characterized by the up......-regulation of several PR genes is notable. Throughout barley embryo development, there are no indications of an increased biosynthesis of either jasmonic acid or salicylic acid. Collectively, the results help explain how the proposed DDA enables protection of the developing barley embryo and grain for purposes...

  15. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  16. Rigorous selection of random forest models for identifying compounds that activate toxicity-related pathways

    Directory of Open Access Journals (Sweden)

    Yoshihiro eUesawa

    2016-02-01

    Full Text Available Random forest (RF is a machine-learning ensemble method with high predictive performance. Majority voting in RF uses the discrimination results in numerous decision trees produced from bootstrapping data. For the same dataset, the bootstrapping process yields different predictive capacities in each generation. As participants in the Toxicology in the 21st Century (Tox21 DATA Challenge 2014, we produced numerous RF models for predicting the structures of compounds that can activate each toxicity-related pathway, and then selected the model with the highest predictive ability. Half of the compounds in the training dataset supplied by the competition organizer were allocated to the validation dataset. The remaining compounds were used in model construction. The charged and uncharged forms of each molecule were calculated using the molecular operating environment (MOE software. Subsequently, the descriptors were computed using MOE, MarvinView, and Dragon. These combined methods yielded over 4,071 descriptors for model construction. Using these descriptors, pattern recognition analyses were performed by RF implemented in JMP Pro (a statistical software package. A hundred to two hundred RF models were generated for each pathway. The predictive performance of each model was tested against the validation dataset, and the best-performing model was selected. In the competition, the latter model selected a best-performing model from the 50% test set that best predicted the structures of compounds that activate the estrogen receptor ligand-binding domain (ER-LBD.

  17. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V;

    2009-01-01

    '-nucleotidase (ecto-5'-NT, CD73), Ndrg1, integrin beta1, CD44, CD74 and MHC class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, immunocyto- and immunohisto-chemistry. Analysis of clinical breast cancer biopsies demonstrated......The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane...

  18. Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma

    OpenAIRE

    Lockwood, William W; Chari, Raj; Coe, Bradley P.; Thu, Kelsie L.; Garnis, Cathie; Malloff, Chad A.; Campbell, Jennifer; Williams, Ariane C.; Hwang, Dorothy; Zhu, Chang-Qi; Buys, Timon P.H.; Yee, John; English, John C.; MacAulay, Calum; Tsao, Ming-sound

    2010-01-01

    Editors' Summary Background Lung cancer is the commonest cause of cancer-related death. Every year, 1.3 million people die from this disease, which is mainly caused by smoking. Most cases of lung cancer are “non-small cell lung cancers” (NSCLCs). Like all cancers, NSCLC starts when cells begin to divide uncontrollably and to move round the body (metastasize) because of changes (mutations) in their genes. These mutations are often in “oncogenes,” genes that, when activated, encourage cell divi...

  19. A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure

    Science.gov (United States)

    Mukhopadhyay, Sayak; Saha, Rohini; Palanisamy, Anbarasi; Ghosh, Madhurima; Biswas, Anupriya; Roy, Saheli; Pal, Arijit; Sarkar, Kathakali; Bagh, Sangram

    2016-05-01

    Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.

  20. A Graph-­based Method to Detect Rare Events: An Application to Identify Pathologic Cells

    OpenAIRE

    Székely, Enikö; Sallaberry, Arnaud; Zaidi, Faraz; Poncelet, Pascal

    2015-01-01

    International audience Detection of outliers and anomalous behavior is a well-­known problem in the data mining and statistics fields. Although the problem of identifying single outliers has been extensively studied in the literature , little or some effort has been devoted to the detection of small groups of outliers that are similar to each other but markedly different from the entire population. Many real world scenarios have small groups of outliers , e. g. a group of students that exc...

  1. >Effect of progesterone hormon on cell viability and stem cell activation in dental pulp cells

    Directory of Open Access Journals (Sweden)

    Segah Altuntaş

    2016-03-01

    Full Text Available Objective: The dental pulp is the part in the center of a tooth made up of living connective tissue and cells called odontoblasts. The vitality of the dentin structure, both during health and after injury, depends on pulp cell activity and the signaling processes that regulate the cell’s behavior. Dental pulp tissue has condensed stem cell activity. Dental pulp stem cells are multipotent stem cells that have the potential to differentiate into a variety of cell types. Several publications have stressed the importance of the expression of pluripotentiality associated markers: the transcription factors Nanog, Sox2, Oct3/4, SSEA4, CD13, Stro1 are indispensable for the stem cells to divide indefinitely without affecting their differentiation potential (self renewal capacity. Progesterone is a steroid hormone leading to menstrual cycle and gestation. There is a widespread rumor among people that pregnancy causes toothy loss. Method: So, progesterone was applied in different concentrations on human dental pulp cells in cell culture. Cell viability assay was applied 24th hour later with trypan blue. RNA isolation, cDNA synthesis and Real Time PCR analysis were applied on selected transcription factors (Nanog and Oct4 (POU5F1 genes which have role on steamness of stem cells. Gene expression analyses results were correlated with the cell viability assay results. Results: Cell viability assay results were 80% viable in control, 82% viable in 7 ml progesterone application, 81% viable in 14 ml progesterone application, 83% viable in 21 ml progesterone application. Due to our findings, progesterone in different concentrations did not chance the cell viability in dental pulpa cells. On gene expression analyses, preliminary results supported that high concentrations of progesterone enhance the gene expressions of steamness genes (Nanog, and Oct4 in dental pulp cells. Conclusions: So, progesterone did not change cell viability in high concentrations. We

  2. High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation.

    Directory of Open Access Journals (Sweden)

    Shakti A Goel

    Full Text Available Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis. Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection. We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization.

  3. Identifying Intracellular Sites of Eicosanoid Lipid Mediator Synthesis with EicosaCell Assays

    OpenAIRE

    Bandeira-Melo, Christianne; Weller, Peter Fahey; Bozza, Patricia T.

    2011-01-01

    Eicosanoids, arachidonic acid-derived signaling lipid mediators, are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. EicosaCell is a microscopic assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eic...

  4. Non-small cell lung cancer in never smokers: a clinical entity to be identified

    Directory of Open Access Journals (Sweden)

    Ilka Lopes Santoro

    2011-01-01

    Full Text Available OBJECTIVES: It has been recognized that patients with non-small cell lung cancer who are lifelong never-smokers constitute a distinct clinical entity. The aim of this study was to assess clinical risk factors for survival among neversmokers with non-small cell lung cancer. METHODS: All consecutive non-small cell lung cancer patients diagnosed (n = 285 between May 2005 and May 2009 were included. The clinical characteristics of never-smokers and ever-smokers (former and current were compared using chi-squared or Student's t tests. Survival curves were calculated using the Kaplan-Meier method, and log-rank tests were used for survival comparisons. A Cox proportional hazards regression analysis was evaluated by adjusting for age (continuous variable, gender (female vs. male, smoking status (never- vs. ever-smoker, the Karnofsky Performance Status Scale (continuous variable, histological type (adenocarcinoma vs. non-adenocarcinoma, AJCC staging (early vs. advanced staging, and treatment (chemotherapy and/or radiotherapy vs. the best treatment support. RESULTS: Of the 285 non-small cell lung cancer patients, 56 patients were never-smokers. Univariate analyses indicated that the never-smoker patients were more likely to be female (68% vs. 32% and have adenocarcinoma (70% vs. 51%. Overall median survival was 15.7 months (95% CI: 13.2 to 18.2. The never-smoker patients had a better survival rate than their counterpart, the ever-smokers. Never-smoker status, higher Karnofsky Performance Status, early staging, and treatment were independent and favorable prognostic factors for survival after adjusting for age, gender, and adenocarcinoma in multivariate analysis. CONCLUSIONS: Epidemiological differences exist between never- and ever-smokers with lung cancer. Overall survival among never-smokers was found to be higher and independent of gender and histological type.

  5. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  6. Automated computation of arbor densities: a step toward identifying neuronal cell types.

    Science.gov (United States)

    Sümbül, Uygar; Zlateski, Aleksandar; Vishwanathan, Ashwin; Masland, Richard H; Seung, H Sebastian

    2014-01-01

    The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference.

  7. Automated computation of arbor densities: a step toward identifying neuronal cell types

    Directory of Open Access Journals (Sweden)

    Uygar eSümbül

    2014-11-01

    Full Text Available The shape and position of a neuron convey information regarding its molecular and functional identity. The identification of cell types from structure, a classic method, relies on the time-consuming step of arbor tracing. However, as genetic tools and imaging methods make data-driven approaches to neuronal circuit analysis feasible, the need for automated processing increases. Here, we first establish that mouse retinal ganglion cell types can be as precise about distributing their arbor volumes across the inner plexiform layer as they are about distributing the skeletons of the arbors. Then, we describe an automated approach to computing the spatial distribution of the dendritic arbors, or arbor density, with respect to a global depth coordinate based on this observation. Our method involves three-dimensional reconstruction of neuronal arbors by a supervised machine learning algorithm, post-processing of the enhanced stacks to remove somata and isolate the neuron of interest, and registration of neurons to each other using automatically detected arbors of the starburst amacrine interneurons as fiducial markers. In principle, this method could be generalizable to other structures of the CNS, provided that they allow sparse labeling of the cells and contain a reliable axis of spatial reference.

  8. Single-Cell Network Analysis Identifies DDIT3 as a Nodal Lineage Regulator in Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Cristina Pina

    2015-06-01

    Full Text Available We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

  9. The invisible addiction: Cell-phone activities and addiction among male and female college students

    Science.gov (United States)

    PETNJI YAYA, LUC HONORE; MANOLIS, CHRIS

    2014-01-01

    Background and aims: The primary objective of the present study was to investigate which cell-phone activities are associated with cell-phone addiction. No research to date has studied the full-range of cell-phone activities, and their relationship to cell-phone addiction, across male and female cell-phone users. Methods: College undergraduates (N = 164) participated in an online survey. Participants completed the questionnaire as part of their class requirements. The questionnaire took 10 and 15 minutes to complete and contained a measure of cell-phone addiction and questions that asked how much time participants spent daily on 24 cell-phone activities. Results: Findings revealed cell-phone activities that are associated significantly with cell-phone addiction (e.g., Instagram, Pinterest), as well as activities that one might logically assume would be associated with this form of addiction but are not (e.g., Internet use and Gaming). Cell-phone activities that drive cell-phone addiction (CPA) were found to vary considerably across male and female cell-phone users. Although a strong social component drove CPA for both males and females, the specific activities associated with CPA differed markedly. Conclusions: CPA amongst the total sample is largely driven by a desire to connect socially. The activities found to be associated with CPA, however, differed across the sexes. As the functionality of cell-phones continues to expand, addiction to this seemingly indispensable piece of technology becomes an increasingly realistic possibility. Future research must identify the activities that push cell-phone use beyond its “;tipping point” where it crosses the line from a helpful tool to one that undermines our personal well-being and that of others. PMID:25595966

  10. Depressed natural killer cell activity in acute myocardial infarction

    DEFF Research Database (Denmark)

    Klarlund, K; Pedersen, B K; Theander, T G;

    1987-01-01

    Natural killer (NK) cell activity against K562 target cells was measured in patients within 24 h of acute myocardial infarction (AMI) and regularly thereafter for 6 weeks. NK cell activity was suppressed on days 1, 3, and 7 (P less than 0.01), day 14 (P less than 0.05) and at 6 weeks (P = 0...

  11. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  12. Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells.

    Science.gov (United States)

    Lefrançais, Emma; Duval, Anais; Mirey, Emilie; Roga, Stéphane; Espinosa, Eric; Cayrol, Corinne; Girard, Jean-Philippe

    2014-10-28

    Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33(95-270), IL-33(107-270), and IL-33(109-270), were 30-fold more potent than full-length human IL-33(1-270) for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33-dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66-111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33-mediated responses in allergic asthma and other inflammatory diseases.

  13. Non-cell autonomous and non-catalytic activities of ATX in the developing brain

    Directory of Open Access Journals (Sweden)

    Raanan eGreenman

    2015-03-01

    Full Text Available The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non cell-autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX as a non cell-autonomous regulator of neural stem cell proliferation. ATX (also known as ENPP2 is best known to catalyze lysophosphatidic acid (LPA production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA.

  14. Thermodynamic Activity Measurements with Knudsen Cell Mass Spectrometry

    Science.gov (United States)

    Copland, Evan H.; Jacobson, Nathan S.

    2001-01-01

    Coupling the Knudsen effusion method with mass spectrometry has proven to be one of the most useful experimental techniques for studying the equilibrium between condensed phases and complex vapors. The Knudsen effusion method involves placing a condensed sample in a Knudsen cell, a small "enclosure", that is uniformly heated and held until equilibrium is attained between the condensed and vapor phases. The vapor is continuously sampled by effusion through a small orifice in the cell. A molecular beam is formed from the effusing vapor and directed into a mass spectrometer for identification and pressure measurement of the species in the vapor phase. Knudsen cell mass spectrometry (KCMS) has been used for nearly fifty years now and continues to be a leading technique for obtaining thermodynamic data. Indeed, much of the well-established vapor specie data in the JANAF tables has been obtained from this technique. This is due to the extreme versatility of the technique. All classes of materials can be studied and all constituents of the vapor phase can be measured over a wide range of pressures (approximately 10(exp -4) to 10(exp -11) bar) and temperatures (500-2800 K). The ability to selectively measure different vapor species makes KCMS a very powerful tool for the measurement of component activities in metallic and ceramic solutions. Today several groups are applying KCMS to measure thermodynamic functions in multicomponent metallic and ceramic systems. Thermodynamic functions, especially component activities, are extremely important in the development of CALPHAD (Calculation of Phase Diagrams) type thermodynamic descriptions. These descriptions, in turn, are useful for modeling materials processing and predicting reactions such as oxide formation and fiber/matrix interactions. The leading experimental methods for measuring activities are the Galvanic cell or electro-motive force (EMF) technique and the KCMS technique. Each has specific advantages, depending on

  15. A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

    Science.gov (United States)

    Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

    2016-01-01

    To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

  16. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

    Institute of Scientific and Technical Information of China (English)

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇

    2004-01-01

    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  17. Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells.

    Science.gov (United States)

    Jaluria, Pratik; Betenbaugh, Michael; Konstantopoulos, Konstantinos; Frank, Bryan; Shiloach, Joseph

    2007-05-01

    The ability to modify cellular properties such as adhesion is of interest in the design and performance of biotechnology-related processes. The current study was undertaken in order to evaluate the effectiveness of modulating cellular adhesion in HeLa cells from a genomics perspective. Using DNA microarrays, differences in gene expression between two phenotypically distinct, anchorage-dependent and anchorage-independent, HeLa cell lines were identified. With the aid of several statistical methods and an extensive literature search, two genes were selected as potential targets for further study: siat7e and lama4. Subsequently, experiments were carried out to investigate the effects of siat7e and lama4 separately, on adhesion in HeLa cells by altering their expression in vivo. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells using short interfering RNA (siRNA) resulted in greater aggregation (i.e. clumping) and morphological changes as compared to untreated anchorage-independent HeLa cells. Similar effects were seen in anchorage-independent HeLa cells when the expression of lama4 which encodes laminin alpha4, a member of the laminin family of glycoproteins, was enhanced as compared to untreated anchorage-independent HeLa cells. Using a shear flow chamber, an attachment assay was developed; illustrating either increased expression of siat7e or decreased expression of lama4 in anchorage-dependent HeLa cells reduced cellular adhesion. Collectively, the results of this study are consistent with the roles siat7e and lama4 play in adhesion processes in vivo and indicate modifying the expression of either gene can influence adhesion in HeLa cells. The strategy of applying bioinformatics techniques to characterize and manipulate phenotypic behaviors is a powerful tool for altering the properties of various cell lines for desired biotechnology objectives. PMID:17240181

  18. Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Sean P Sherman

    Full Text Available Differentiated cells from human embryonic stem cells (hESCs provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

  19. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    Science.gov (United States)

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

  20. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    Science.gov (United States)

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients. PMID:27019998

  1. Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

    OpenAIRE

    Wallace, Jared; Hu, Ruozhen; Mosbruger, Timothy L.; Dahlem, Timothy J.; Stephens, W. Zac; Rao, Dinesh S.; Round, June L.; O’Connell, Ryan M.

    2016-01-01

    Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that m...

  2. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    Science.gov (United States)

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  3. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration

    International Nuclear Information System (INIS)

    Mitochondrial impairment is increasingly implicated in the etiology of toxicity caused by some thiazolidinediones, fibrates, and statins. We examined the effects of members of these drug classes on respiration of isolated rat liver mitochondria using a phosphorescent oxygen sensitive probe and on the activity of individual oxidative phosphorylation (OXPHOS) complexes using a recently developed immunocapture technique. Of the six thiazolidinediones examined, ciglitazone, troglitazone, and darglitazone potently disrupted mitochondrial respiration. In accord with these data, ciglitazone and troglitazone were also potent inhibitors of Complexes II + III, IV, and V, while darglitazone predominantly inhibited Complex IV. Of the six statins evaluated, lovastatin, simvastatin, and cerivastatin impaired mitochondrial respiration the most, with simvastatin and lovastatin impairing multiple OXPHOS Complexes. Within the class of fibrates, gemfibrozil more potently impaired respiration than fenofibrate, clofibrate, or ciprofibrate. Gemfibrozil only modestly inhibited Complex I, fenofibrate inhibited Complexes I, II + III, and V, and clofibrate inhibited Complex V. Our findings with the two complementary methods indicate that (1) some members of each class impair mitochondrial respiration, whereas others have little or no effect, and (2) the rank order of mitochondrial impairment accords with clinical adverse events observed with these drugs. Since the statins are frequently co-prescribed with the fibrates or thiazolidinediones, various combinations of these three drug classes were also analyzed for their mitochondrial effects. In several cases, the combination additively uncoupled or inhibited respiration, suggesting that some combinations are more likely to yield clinically relevant drug-induced mitochondrial side effects than others

  4. Neutron activation analysis techniques for identifying elemental status in Alzheimer's disease

    International Nuclear Information System (INIS)

    Brain tissue (hippocampus and cerebral cortex) from Alzheimer's disease and control individuals sampled from Eastern Canada and the United Kingdom were analyzed for Ag, Al, As, B, Br, Ca, Cd, Co, Cr, Cs, Cu, Fe, Hg, I, K, La, Mg, Mn, Mo, Ni, Rb, S, Sb, Sc, Se, Si, Sn, Sr, Ti, V and Zn. Neutron activation analysis (thermal and prompt gamma-ray) methods were used. Very highly significant differences (S**: probability less than 0.005) for both study areas were shown between Alzheimer's disease (AD) and control (C) individuals: AD>C for Al, Br, Ca and S, and AD< C for Se, V and Zn. Aluminium content of brain tissue ranged form 3.605 to 21.738 μg/g d.w. (AD) and 0.379 to 4.768 μg/g d.w. (C). No statistical evidence of aluminium accumulation with age was noted. Possible zinc deficiency (especially for hippocampal tissue), was observed with zinc ranges of 31.42 to 57.91 μg/g d.w. (AD) and 37.31 to 87.10 μg/g d.w. (C), for Alzheimer's disease patients. (author)

  5. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    Science.gov (United States)

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  6. Myeloid-derived Suppressor Cells Inhibit T Cell Activation by Depleting Cystine and Cysteine

    OpenAIRE

    Minu K Srivastava; Sinha, Pratima; Clements, Virginia K.; Rodriguez, Paulo; Ostrand-Rosenberg, Suzanne

    2009-01-01

    Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated anti-tumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and IL-10. We now report that MDSC also block T cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T cell activation because T cells lack cystathionase, which...

  7. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    OpenAIRE

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Richard K Assoian; Rader, Daniel J; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD4...

  8. Gene expression analysis of PTEN positive glioblastoma stem cells identifies DUB3 and Wee1 modulation in a cell differentiation model.

    Directory of Open Access Journals (Sweden)

    Stefano Forte

    Full Text Available The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.

  9. A cell-based in vitro alternative to identify skin sensitizers by gene expression

    International Nuclear Information System (INIS)

    The ethical and economic burden associated with animal testing for assessment of skin sensitization has triggered intensive research effort towards development and validation of alternative methods. In addition, new legislation on the registration and use of cosmetics and chemicals promote the use of suitable alternatives for hazard assessment. Our previous studies demonstrated that human CD34+ progenitor-derived dendritic cells from cord blood express specific gene profiles upon exposure to low molecular weight sensitizing chemicals. This paper presents a classification model based on this cell type which is successful in discriminating sensitizing chemicals from non-sensitizing chemicals based on transcriptome analysis of 13 genes. Expression profiles of a set of 10 sensitizers and 11 non-sensitizers were analyzed by RT-PCR using 9 different exposure conditions and a total of 73 donor samples. Based on these data a predictive dichotomous classifier for skin sensitizers has been constructed, which is referred to as . In a first step the dimensionality of the input data was reduced by selectively rejecting a number of exposure conditions and genes. Next, the generalization of a linear classifier was evaluated by a cross-validation which resulted in a prediction performance with a concordance of 89%, a specificity of 97% and a sensitivity of 82%. These results show that the present model may be a useful human in vitro alternative for further use in a test strategy towards the reduction of animal use for skin sensitization

  10. A tenascin-C aptamer identified by tumor cell SELEX: Systematic evolution of ligands by exponential enrichment

    Science.gov (United States)

    Daniels, Dion A.; Chen, Hang; Hicke, Brian J.; Swiderek, Kristine M.; Gold, Larry

    2003-01-01

    The targeting of molecular repertoires to complex systems rather than biochemically pure entities is an accessible approach that can identify proteins of biological interest. We have probed antigens presented by a monolayer of tumor cells for their ability to interact with a pool of aptamers. A glioblastoma-derived cell line, U251, was used as the target for systematic evolution of ligands by exponential enrichment by using a single-stranded DNA library. We isolated specifically interacting oligonucleotides, and biochemical strategies were used to identify the protein target for one of the aptamers. Here we characterize the interaction of the DNA aptamer, GBI-10, with tenascin-C, an extracellular protein found in the tumor matrix. Tenascin-C is believed to be involved in both embryogenesis and oncogenesis pathways. Systematic evolution of ligands by exponential enrichment appears to be a successful strategy for the a priori identification of targets of biological interest within complex systems. PMID:14676325

  11. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  12. Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells.

    Directory of Open Access Journals (Sweden)

    Henry Haeberle

    Full Text Available Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute

  13. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome

    Science.gov (United States)

    Harlow, Philippa H.; Perry, Simon J.; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A.; Flemming, Anthony J.

    2016-01-01

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals. PMID:26987796

  14. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.

    Science.gov (United States)

    Harlow, Philippa H; Perry, Simon J; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A; Flemming, Anthony J

    2016-01-01

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

  15. Teachers' instructional goals for science practice: Identifying knowledge gaps using cultural-historical activity theory (CHAT)

    Science.gov (United States)

    Farrar, Cynthia Hamen

    In AP Biology, the course goal, with respect to scientific acts and reasoning, has recently shifted toward a reform goal of science practice, where the goal is for students to have a scientific perspective that views science as a practice of a community rather than a body of knowledge. Given this recent shift, this study is interested in the gaps that may exist between an individual teacher's instructional goal and the goals of the AP Biology course. A Cultural-Historical Activity Theory (CHAT) methodology and perspective is used to analyze four teachers' knowledge, practice, and learning. Teachers have content knowledge for teaching, a form of knowledge that is unique for teaching called specialized content knowledge. This specialized content knowledge (SCK) defines their instructional goals, the student outcomes they ultimately aim to achieve with their students. The study employs a cultural-historical continuum of scientific acts and reasoning, which represents the development of the AP Biology goal over time, to study gaps in their instructional goal. The study also analyzes the contradictions within their teaching practice and how teachers address those contradictions to shift their instructional practice and learn. The findings suggest that teachers have different interpretations of the AP Biology goals of science practice, placing their instructional goal at different points along the continuum. Based on the location of their instructional goal, different micro-communities of teachers exist along the continuum, comprised of teachers with a shared goal, language, and culture of their AP Biology teaching. The in-depth study of one teacher's AP Biology teaching, using a CHAT perspective, provides a means for studying the mechanisms that connect SCK to classroom actions and ultimately to instructional practice. CHAT also reveals the nature and importance of contradictions or cognitive dissonance in teacher learning and the types of support teachers need to

  16. Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

    Directory of Open Access Journals (Sweden)

    Christopher Vollmers

    Full Text Available Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

  17. Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier Transform Near Infrared spectroscopy

    Directory of Open Access Journals (Sweden)

    Smith-Moritz Andreia M

    2011-08-01

    Full Text Available Abstract We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.

  18. Identifying subgroups among poor prognosis patients with nonseminomatous germ cell cancer by tree modelling: a validation study.

    OpenAIRE

    Dijk, Merel; Steyerberg, Ewout; Stenning, S. P.; Habbema, Dik

    2004-01-01

    textabstractBACKGROUND: In order to target intensive treatment strategies for poor prognosis patients with non-seminomatous germ cell cancer, those with the poorest prognosis should be identified. These patients might profit most from more intensive treatment strategies. For this purpose, a regression tree was previously developed on 332 patients. We aimed to evaluate the performance and structure of this tree. PATIENTS AND METHODS: The previously developed tree was applied to 456 patients wi...

  19. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    Science.gov (United States)

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  20. Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

    International Nuclear Information System (INIS)

    To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle

  1. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    DEFF Research Database (Denmark)

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen;

    2015-01-01

    densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  2. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  3. Interleukin 4 (B cell stimulatory factor 1) can mediate the induction of lymphokine-activated killer cell activity directed against fresh tumor cells

    OpenAIRE

    1987-01-01

    Interleukin 4 (IL-4) expresses multiple biologic activities, including B cell, mast cell, and T cell stimulation. We showed that the incubation of resting splenocytes from C57BL/6 mice solely in purified native or recombinant mouse IL-4 results in the generation of lymphokine-activated killer (LAK) activity directed against fresh, syngeneic sarcoma cells. The precursor activated by IL-4 expresses surface asialo-GM1. In addition, IL-4 is capable of amplifying the splenic LAK activity induced b...

  4. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    International Nuclear Information System (INIS)

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic 75selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by 51Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances

  5. Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins.

    Science.gov (United States)

    Wang, Yang; Yi, Li; Wu, Zongfu; Shao, Jing; Liu, Guangjin; Fan, Hongjie; Zhang, Wei; Lu, Chengping

    2012-01-01

    Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.

  6. Tolerogenicity of resting and activated B cells

    OpenAIRE

    1994-01-01

    Antigen presentation by resting splenic B cells has been shown previously to induce T helper 1 cell (Th1) anergy. In contrast to expectations, it was found here that B cells treated with F(ab')2 goat anti-mouse immunoglobulin (IgM) for 24 or 48 h also presented antigen (Ag) to Th1 cells in a manner that induced dramatic Ag-specific proliferative inactivation. The tolerogenicity of the anti-Ig-treated B cells was consistent with the observation that these B cells were only slightly more effici...

  7. Optimization and experimental validation of a thermal cycle that maximizes entropy coefficient fisher identifiability for lithium iron phosphate cells

    Science.gov (United States)

    Mendoza, Sergio; Rothenberger, Michael; Hake, Alison; Fathy, Hosam

    2016-03-01

    This article presents a framework for optimizing the thermal cycle to estimate a battery cell's entropy coefficient at 20% state of charge (SOC). Our goal is to maximize Fisher identifiability: a measure of the accuracy with which a parameter can be estimated. Existing protocols in the literature for estimating entropy coefficients demand excessive laboratory time. Identifiability optimization makes it possible to achieve comparable accuracy levels in a fraction of the time. This article demonstrates this result for a set of lithium iron phosphate (LFP) cells. We conduct a 24-h experiment to obtain benchmark measurements of their entropy coefficients. We optimize a thermal cycle to maximize parameter identifiability for these cells. This optimization proceeds with respect to the coefficients of a Fourier discretization of this thermal cycle. Finally, we compare the estimated parameters using (i) the benchmark test, (ii) the optimized protocol, and (iii) a 15-h test from the literature (by Forgez et al.). The results are encouraging for two reasons. First, they confirm the simulation-based prediction that the optimized experiment can produce accurate parameter estimates in 2 h, compared to 15-24. Second, the optimized experiment also estimates a thermal time constant representing the effects of thermal capacitance and convection heat transfer.

  8. Identifying carcinogenic activity of methylated and non-methylated polycyclic aromatic hydrocarbons (PAHs) through electronic and topological indices

    CERN Document Server

    Braga, R S; Barone, P M V B

    2000-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are a class of planar molecules, abundant in urban environment, which can induce chemical carcinogenesis. Their carcinogenic power varies in a large range, from very strong carcinogens to inactive ones. In a previous study, we proposed a methodology to identify the PAHs carcinogenic activity exploring electronic and topological indices. In the present work, we show that it is possible to simplify that methodology and expand its applicability to include methylated PAHs compounds. Using very simple rules, we can predict their carcinogenic activity with high accuracy (approx 89%).

  9. Gene Expression Profiling Identifies Cell Proliferation and Inflammation as the Predominant Pathways Regulated by Aryl Hydrocarbon Receptor in Primary Human Fetal Lung Cells Exposed to Hyperoxia.

    Science.gov (United States)

    Shivanna, Binoy; Maity, Suman; Zhang, Shaojie; Patel, Ananddeep; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E; Belmont, John; Coarfa, Cristian; Moorthy, Bhagavatula

    2016-07-01

    Exposure to hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. We observed that aryl hydrocarbon receptor (AhR) signaling protects newborn mice and primary fetal human pulmonary microvascular endothelial cells (HPMECs) against hyperoxic injury. Additionally, a recent genome-wide transcriptome study in a newborn mouse model of BPD identified AhR as a key regulator of hyperoxia-induced gene dysregulation. Whether the AhR similarly deregulates genes in HPMEC is unknown. Therefore, the objective of this study was to characterize transcriptome level gene expression profile in AhR-sufficient and -deficient HPMEC exposed to normoxic and hyperoxic conditions. Global gene expression profiling was performed using Illumina microarray platform and selected genes were validated by real-time RT-PCR. AhR gene expression and hyperoxia independently affected the expression of 540 and 593 genes, respectively. Two-way ANOVA further identified 85 genes that were affected by an interaction between AhR expression and exposure to hyperoxia. Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology, and Reactome pathway analysis identified cell proliferation, immune function, cytokine signaling, and organ development as the major pathways affected in AhR-deficient cells. The biological processes that were significantly enriched by hyperoxia included metabolic process, stress response, signal transduction, cell cycle, and immune regulation. Cell cycle was the predominant pathway affected by the combined effect of AhR knockdown and hyperoxia. Functional analysis of cell cycle showed that AhR-deficient cells had decreased proliferation compared with AhR-sufficient cells. These findings suggest that AhR modulates hyperoxic lung injury by regulating the genes that are necessary for cell proliferation and inflammation. PMID:27103661

  10. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    Science.gov (United States)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  11. Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

    Directory of Open Access Journals (Sweden)

    Lu Fachuang

    2010-06-01

    Full Text Available Abstract Background Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production. Results In the absence of anatomical constraints to digestion, lignification with normal monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree

  12. Novel curcumin- and emodin-related compounds identified by in silico 2D/3D conformer screening induce apoptosis in tumor cells

    International Nuclear Information System (INIS)

    Inhibition of the COP9 signalosome (CSN) associated kinases CK2 and PKD by curcumin causes stabilization of the tumor suppressor p53. It has been shown that curcumin induces tumor cell death and apoptosis. Curcumin and emodin block the CSN-directed c-Jun signaling pathway, which results in diminished c-Jun steady state levels in HeLa cells. The aim of this work was to search for new CSN kinase inhibitors analogue to curcumin and emodin by means of an in silico screening method. Here we present a novel method to identify efficient inhibitors of CSN-associated kinases. Using curcumin and emodin as lead structures an in silico screening with our in-house database containing more than 106 structures was carried out. Thirty-five compounds were identified and further evaluated by the Lipinski's rule-of-five. Two groups of compounds can be clearly discriminated according to their structures: the curcumin-group and the emodin-group. The compounds were evaluated in in vitro kinase assays and in cell culture experiments. The data revealed 3 compounds of the curcumin-group (e.g. piceatannol) and 4 of the emodin-group (e.g. anthrachinone) as potent inhibitors of CSN-associated kinases. Identified agents increased p53 levels and induced apoptosis in tumor cells as determined by annexin V-FITC binding, DNA fragmentation and caspase activity assays. Our data demonstrate that the new in silico screening method is highly efficient for identifying potential anti-tumor drugs

  13. Single-Cell, Genome-wide Sequencing Identifies Clonal Somatic Copy-Number Variation in the Human Brain

    Directory of Open Access Journals (Sweden)

    Xuyu Cai

    2014-09-01

    Full Text Available De novo copy-number variants (CNVs can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥95% neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ∼20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains.

  14. Identifying type 1 diabetes candidate genes by DNA microarray analysis of islet-specific CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Gregory J. Berry

    2015-09-01

    Full Text Available Type 1 diabetes (T1D is a T cell-mediated autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells and is fatal unless treated with insulin. During the last four decades, multiple insulin-dependent diabetes (Idd susceptibility/resistance loci that regulate T1D development have been identified in humans and non-obese diabetic (NOD mice, an established animal model for T1D. However, the exact mechanisms by which these loci confer diabetes risk and the identity of the causative genes remain largely elusive. To identify genes and molecular mechanisms that control the function of diabetogenic T cells, we conducted DNA microarray analysis in islet-specific CD4+ T cells from BDC2.5 TCR transgenic NOD mice that contain the Idd9 locus from T1D-susceptible NOD mice or T1D-resistant C57BL/10 mice. Here we describe in detail the contents and analyses for these gene expression data associated with our previous study [1]. Gene expression data are available at the Gene Expression Omnibus (GEO repository from the National Center for Biotechnology Information (accession number GSE64674.

  15. Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Oliver Birkholz

    2015-03-01

    Full Text Available The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS of Drosophila, neural stem cells (neuroblasts exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

  16. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53

    Directory of Open Access Journals (Sweden)

    Arindam Datta

    2016-06-01

    Full Text Available Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR treated SW480 cells expressing mutant p53R273H (GEO#: GSE77533. We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.

  17. Cell-Specific mRNA Profiling of the Caenorhabditis elegans Somatic Gonadal Precursor Cells Identifies Suites of Sex-Biased and Gonad-Enriched Transcripts.

    Science.gov (United States)

    Kroetz, Mary B; Zarkower, David

    2015-12-01

    The Caenorhabditis elegans somatic gonad differs greatly between the two sexes in its pattern of cell divisions, migration, and differentiation. Despite decades of study, the genetic pathways directing early gonadal development and establishing sexual dimorphism in the gonad remain largely unknown. To help define the genetic networks that regulate gonadal development, we employed cell-specific RNA-seq. We identified transcripts present in the somatic gonadal precursor cells and their daughter cells of each sex at the onset of sexual differentiation. We identified several hundred gonad-enriched transcripts, including the majority of known regulators of early gonadal development, and transgenic reporter analysis confirmed the effectiveness of this approach. Before the division of the somatic gonad precursors, few sex-biased gonadal transcripts were detectable; less than 6 hr later, after their division, we identified more than 250 sex-biased transcripts, of which about a third were enriched in the somatic gonad compared to the whole animal. This indicates that a robust sex-biased developmental program, some of it gonad-specific, initiates in the somatic gonadal precursor cells around the time of their first division. About 10% of male-biased transcripts had orthologs with male-biased expression in the early mouse gonad, suggesting possible conservation of gonad sex differentiation. Cell-specific analysis also identified approximately 70 previously unannotated mRNA isoforms that are enriched in the somatic gonad. Our data illustrate the power of cell-specific transcriptome analysis and suggest that early sex differentiation in the gonad is controlled by a relatively small suite of differentially expressed genes, even after dimorphism has become apparent. PMID:26497144

  18. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Science.gov (United States)

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  19. Larvicidal activity of medicinal plant extracts and lignan identified in Phryma leptostachya var. asiatica roots against housefly (Musca domestica L.).

    Science.gov (United States)

    Seo, Seon-Mi; Park, Il-Kwon

    2012-05-01

    Medicinal plant extracts from 27 plant species in 20 families were tested for their larvicidal activity against housefly, Musca domestica (L.). Responses varied with plant material and concentration. Among plant species tested, Phryma leptostachya var. asiatica showed 100% larvicidal activity against M. domestica at 10 mg/g concentration. Larvicidal activities of Atractylodes japonica, Saussurea lappa, Asiasarum sieboldi, and Gleditsia japonica var. koraiensis were 89.3%, 85.3%, 93.3%, and 96.6% at 10 mg/g concentration, respectively. Extracts of Prunus persica, Curcuma longa, and Paeonia moutan produced moderate activity. Larvicidal activity of other plant extracts was less than 50%. Among test plant species, P. leptostachya var. asiatica showed the most potent larvicidal activity. The active constituent of P. leptostachya var. asiatica roots was identified as the leptostachyol acetate by spectroscopic analysis. The LC(50) values of leptostachyol acetate against M. domestica larvae were 0.039 mg/g. Naturally occurring medicinal plant extracts and P. leptostachya var. asiatica root-derived compounds merit further study as potential housefly larval control agents or lead compounds.

  20. Signal integration by Ca2+ regulates intestinal stem cell activity

    Science.gov (United States)

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  1. Asymptomatic Multiple Lymphomatous Polyposis Identified during Staging Bidirectional Endoscopy of Mantle Cell Lymphoma

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    Sonja P. Dawsey

    2016-10-01

    Full Text Available Multiple lymphomatous polyposis (MLP as an extranodal manifestation of mantle cell lymphoma (MCL in the gastrointestinal tract is rare and not often reported in the literature. We describe the case of a 63-year-old female with asymptomatic MLP found during staging bidirectional endoscopy of MCL. The patient presented only with dyspnea, but was found on physical exam to have diffuse lymphadenopathy, and subsequent positron emission tomography (PET CT showed extensive lymph node adenopathy consistent with lymphoma. Excisional lymph node biopsy revealed high-risk MCL. Prior to therapy, staging bidirectional endoscopy was performed, which revealed duodenal bulb polyps and diffuse polyposis in the colon. Biopsies showed atypical lymphoid infiltrate identical to the initial excisional lymph node biopsy. The patient underwent aggressive induction therapy, chemotherapy and bone marrow transplantation. Four months later, repeat colonoscopy and biopsies showed normal mucosa, and repeat PET CT showed no evidence of systemic disease. Eight months later, the patient began having symptoms consistent with cauda equina syndrome, and she was found to have leptomeningeal recurrence of MCL. In spite of other medical treatment, the patient’s MCL progressed and she passed away 3 years after the initial presentation.

  2. Remote Control of T Cell Activation Using Magnetic Janus Particles.

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    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  3. Microbial fuel cells with highly active aerobic biocathodes

    Science.gov (United States)

    Milner, Edward M.; Popescu, Dorin; Curtis, Tom; Head, Ian M.; Scott, Keith; Yu, Eileen H.

    2016-08-01

    Microbial fuel cells (MFCs), which convert organic waste to electricity, could be used to make the wastewater infrastructure more energy efficient and sustainable. However, platinum and other non-platinum chemical catalysts used for the oxygen reduction reaction (ORR) at the cathode of MFCs are unsustainable due to their high cost and long-term degradation. Aerobic biocathodes, which use microorganisms as the biocatalysts for cathode ORR, are a good alternative to chemical catalysts. In the current work, high-performing aerobic biocathodes with an onset potential for the ORR of +0.4 V vs. Ag/AgCl were enriched from activated sludge in electrochemical half-cells poised at -0.1 and + 0.2 V vs. Ag/AgCl. Gammaproteobacteria, distantly related to any known cultivated gammaproteobacterial lineage, were identified as dominant in these working electrode biofilms (23.3-44.3% of reads in 16S rRNA gene Ion Torrent libraries), and were in very low abundance in non-polarised control working electrode biofilms (0.5-0.7%). These Gammaproteobacteria were therefore most likely responsible for the high activity of biologically catalysed ORR. In MFC tests, a high-performing aerobic biocathode increased peak power 9-fold from 7 to 62 μW cm-2 in comparison to an unmodified carbon cathode, which was similar to peak power with a platinum-doped cathode at 70 μW cm-2.

  4. Procalcitonin Identifies Cell Injury, Not Bacterial Infection, in Acute Liver Failure.

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    Jody A Rule

    Full Text Available Because acute liver failure (ALF patients share many clinical features with severe sepsis and septic shock, identifying bacterial infection clinically in ALF patients is challenging. Procalcitonin (PCT has proven to be a useful marker in detecting bacterial infection. We sought to determine whether PCT discriminated between presence and absence of infection in patients with ALF.Retrospective analysis of data and samples of 115 ALF patients from the United States Acute Liver Failure Study Group randomly selected from 1863 patients were classified for disease severity and ALF etiology. Twenty uninfected chronic liver disease (CLD subjects served as controls.Procalcitonin concentrations in most samples were elevated, with median values for all ALF groups near or above a 2.0 ng/mL cut-off that generally indicates severe sepsis. While PCT concentrations increased somewhat with apparent liver injury severity, there were no differences in PCT levels between the pre-defined severity groups-non-SIRS and SIRS groups with no documented infections and Severe Sepsis and Septic Shock groups with documented infections, (p = 0.169. PCT values from CLD patients differed from all ALF groups (median CLD PCT value 0.104 ng/mL, (p ≤0.001. Subjects with acetaminophen (APAP toxicity, many without evidence of infection, demonstrated median PCT >2.0 ng/mL, regardless of SIRS features, while some culture positive subjects had PCT values <2.0 ng/mL.While PCT appears to be a robust assay for detecting bacterial infection in the general population, there was poor discrimination between ALF patients with or without bacterial infection presumably because of the massive inflammation observed. Severe hepatocyte necrosis with inflammation results in elevated PCT levels, rendering this biomarker unreliable in the ALF setting.