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Sample records for cells reveals roles

  1. Optogenetic toolkit reveals the role of Ca2+ sparklets in coordinated cell migration.

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    Kim, Jin Man; Lee, Minji; Kim, Nury; Heo, Won Do

    2016-05-24

    Cell migration is controlled by various Ca(2+) signals. Local Ca(2+) signals, in particular, have been identified as versatile modulators of cell migration because of their spatiotemporal diversity. However, little is known about how local Ca(2+) signals coordinate between the front and rear regions in directionally migrating cells. Here, we elucidate the spatial role of local Ca(2+) signals in directed cell migration through combinatorial application of an optogenetic toolkit. An optically guided cell migration approach revealed the existence of Ca(2+) sparklets mediated by L-type voltage-dependent Ca(2+) channels in the rear part of migrating cells. Notably, we found that this locally concentrated Ca(2+) influx acts as an essential transducer in establishing a global front-to-rear increasing Ca(2+) gradient. This asymmetrical Ca(2+) gradient is crucial for maintaining front-rear morphological polarity by restricting spontaneous lamellipodia formation in the rear part of migrating cells. Collectively, our findings demonstrate a clear link between local Ca(2+) sparklets and front-rear coordination during directed cell migration.

  2. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

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    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  3. Role of SLAM in NKT cell development revealed by transgenic complementation in NOD mice.

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    Jordan, Margaret A; Fletcher, Julie M; Jose, Roby; Chowdhury, Shahead; Gerlach, Nicole; Allison, Janette; Baxter, Alan G

    2011-04-01

    Allelic variation of SLAM expression on CD4(+)CD8(+) thymocytes has been proposed to play a major role in NKT cell development. In this article, this hypothesis is tested by the production of subcongenic mouse strains and Slamf1 transgenic lines. The long isoform of the C57BL/6 allele of Slamf1 was transgenically expressed on CD4(+)CD8(+) thymocytes under control of an hCD2 minigene. NOD.Nkrp1b.Tg(Slamf1)1 mice, which had a 2-fold increase in SLAM protein expression on CD4(+)CD8(+) thymocytes, had a 2-fold increase in numbers of thymic NKT cells. The additional thymic NKT cells in NOD.Nkrp1b.Tg(Slamf1)1 mice were relatively immature, with a similar subset distribution to those of congenic NOD.Nkrp1b.Nkt1 and NOD.Nkrp1b.Slamf1 mice, which also express increased levels of SLAM on CD4(+)CD8(+) thymocytes and produce larger numbers of NKT cells. Transgenic enhancement of SLAM expression also increased IL-4 and IL-17 production in response to TCR-mediated stimulation. Paradoxically, NOD.Nkrp1b.Tg(Slamf1)2 mice, which had a 7-fold increase in SLAM expression, showed no significant increase in NKT cells numbers; on the contrary, at high transgene copy number, SLAM expression levels correlated inversely with NKT cell numbers, consistent with a contribution to negative selection. These data confirm a role for SLAM in controlling NKT cell development and are consistent with a role in both positive and negative thymic selection of NKT cells.

  4. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

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    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-05-07

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

  5. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease

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    Natalia Rodriguez-Muela

    2017-02-01

    Full Text Available The mechanism underlying selective motor neuron (MN death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.

  6. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

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    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  7. Stem cell heterogeneity revealed

    DEFF Research Database (Denmark)

    Andersen, Marianne S; Jensen, Kim B

    2016-01-01

    The skin forms a protective, water-impermeable barrier consisting of heavily crosslinked epithelial cells. However, the specific role of stem cells in sustaining this barrier remains a contentious issue. A detailed analysis of the interfollicular epidermis now proposes a model for how a composite...... of cells with different properties are involved in its maintenance....

  8. Chemotaxis: new role for Ras revealed

    Institute of Scientific and Technical Information of China (English)

    Jianshe Yan; Dale Hereld; Tian Jin

    2010-01-01

    @@ A recent study of chemotaxis revealed a new role for the proto-oncogene Ras in the social ameba Dictyostelium discoideum.Chemotaxis,the directional movement of cells toward chemokines and other chemoattractants,plays critical roles in diverse physiological processes,such as mobilization of immune cells to fight invading microorganisms,targeting of metastatic cancer cells to specific tissues,and guidance of sperm cells to ova during fertilization.This work,published in the July 26 issue of The Journal of Cell Biology,was conducted in Dr.Devreotes' lab at John Hopkins University and Dr.Parent's lab at National Cancer Institute.This research team demonstrated that RasC functions as an upstream regulator of TORC2 and thereby governs the effects of TORC2-PKB signaling on the cytoskeleton and cell migration.

  9. A Stochastic Model of the Yeast Cell Cycle Reveals Roles for Feedback Regulation in Limiting Cellular Variability.

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    Barik, Debashis; Ball, David A; Peccoud, Jean; Tyson, John J

    2016-12-01

    The cell division cycle of eukaryotes is governed by a complex network of cyclin-dependent protein kinases (CDKs) and auxiliary proteins that govern CDK activities. The control system must function reliably in the context of molecular noise that is inevitable in tiny yeast cells, because mistakes in sequencing cell cycle events are detrimental or fatal to the cell or its progeny. To assess the effects of noise on cell cycle progression requires not only extensive, quantitative, experimental measurements of cellular heterogeneity but also comprehensive, accurate, mathematical models of stochastic fluctuations in the CDK control system. In this paper we provide a stochastic model of the budding yeast cell cycle that accurately accounts for the variable phenotypes of wild-type cells and more than 20 mutant yeast strains simulated in different growth conditions. We specifically tested the role of feedback regulations mediated by G1- and SG2M-phase cyclins to minimize the noise in cell cycle progression. Details of the model are informed and tested by quantitative measurements (by fluorescence in situ hybridization) of the joint distributions of mRNA populations in yeast cells. We use the model to predict the phenotypes of ~30 mutant yeast strains that have not yet been characterized experimentally.

  10. Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8(+) T cells.

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    Wen, Jinsheng; Tang, William Weihao; Sheets, Nicholas; Ellison, Julia; Sette, Alessandro; Kim, Kenneth; Shresta, Sujan

    2017-03-13

    CD8(+) T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/β receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-γ-ELISPOT and IFN-γ-ICS on CD8(+) T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8(+) T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8(+) T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8(+) T cell responses that reduced infectious ZIKV levels, and CD8(+) T cell depletions confirmed that CD8(+) T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8(+) T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8(+) T cell responses of human relevance.

  11. Polynomial algebra reveals diverging roles of the unfolded protein response in endothelial cells during ischemia-reperfusion injury.

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    Le Pape, Sylvain; Dimitrova, Elena; Hannaert, Patrick; Konovalov, Alexander; Volmer, Romain; Ron, David; Thuillier, Raphaël; Hauet, Thierry

    2014-08-25

    The unfolded protein response (UPR)--the endoplasmic reticulum stress response--is found in various pathologies including ischemia-reperfusion injury (IRI). However, its role during IRI is still unclear. Here, by combining two different bioinformatical methods--a method based on ordinary differential equations (Time Series Network Inference) and an algebraic method (probabilistic polynomial dynamical systems)--we identified the IRE1α-XBP1 and the ATF6 pathways as the main UPR effectors involved in cell's adaptation to IRI. We validated these findings experimentally by assessing the impact of their knock-out and knock-down on cell survival during IRI.

  12. Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

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    He Xianghuo

    2010-07-01

    Full Text Available Abstract Background The formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms. Methods The human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis. Results A spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line. Conclusions We have successfully established a new human lung cancer cell line with

  13. Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

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    Duncan, S A

    2005-12-01

    Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

  14. Molecular Characterization of Down Syndrome Embryonic Stem Cells Reveals a Role for RUNX1 in Neural Differentiation

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    Tomer Halevy

    2016-10-01

    Full Text Available Down syndrome (DS is the leading genetic cause of mental retardation and is caused by a third copy of human chromosome 21. The different pathologies of DS involve many tissues with a distinct array of neural phenotypes. Here we characterize embryonic stem cell lines with DS (DS-ESCs, and focus on the neural aspects of the disease. Our results show that neural progenitor cells (NPCs differentiated from five independent DS-ESC lines display increased apoptosis and downregulation of forehead developmental genes. Analysis of differentially expressed genes suggested RUNX1 as a key transcription regulator in DS-NPCs. Using genome editing we were able to disrupt all three copies of RUNX1 in DS-ESCs, leading to downregulation of several RUNX1 target developmental genes accompanied by reduced apoptosis and neuron migration. Our work sheds light on the role of RUNX1 and the importance of dosage balance in the development of neural phenotypes in DS.

  15. Modeling human retinal development with patient-specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2.

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    Phillips, M Joseph; Perez, Enio T; Martin, Jessica M; Reshel, Samantha T; Wallace, Kyle A; Capowski, Elizabeth E; Singh, Ruchira; Wright, Lynda S; Clark, Eric M; Barney, Patrick M; Stewart, Ron; Dickerson, Sarah J; Miller, Michael J; Percin, E Ferda; Thomson, James A; Gamm, David M

    2014-06-01

    Human induced pluripotent stem cells (hiPSCs) have been shown to differentiate along the retinal lineage in a manner that mimics normal mammalian development. Under certain culture conditions, hiPSCs form optic vesicle-like structures (OVs), which contain proliferating progenitors capable of yielding all neural retina (NR) cell types over time. Such observations imply conserved roles for regulators of retinogenesis in hiPSC-derived cultures and the developing embryo. However, whether and to what extent this assumption holds true has remained largely uninvestigated. We examined the role of a key NR transcription factor, visual system homeobox 2 (VSX2), using hiPSCs derived from a patient with microphthalmia caused by an R200Q mutation in the VSX2 homeodomain region. No differences were noted between (R200Q)VSX2 and sibling control hiPSCs prior to OV generation. Thereafter, (R200Q)VSX2 hiPSC-OVs displayed a significant growth deficit compared to control hiPSC-OVs, as well as increased production of retinal pigmented epithelium at the expense of NR cell derivatives. Furthermore, (R200Q)VSX2 hiPSC-OVs failed to produce bipolar cells, a distinctive feature previously observed in Vsx2 mutant mice. (R200Q)VSX2 hiPSC-OVs also demonstrated delayed photoreceptor maturation, which could be overcome via exogenous expression of wild-type VSX2 at early stages of retinal differentiation. Finally, RNAseq analysis on isolated hiPSC-OVs implicated key transcription factors and extracellular signaling pathways as potential downstream effectors of VSX2-mediated gene regulation. Our results establish hiPSC-OVs as versatile model systems to study retinal development at stages not previously accessible in humans and support the bona fide nature of hiPSC-OV-derived retinal progeny.

  16. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

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    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  17. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid.

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    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51-64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic.

  18. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

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    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  19. Cell type-specific transcriptome analysis reveals a major role for Zeb1 and miR-200b in mouse inner ear morphogenesis.

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    Ronna Hertzano

    2011-09-01

    Full Text Available Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type-specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants.

  20. Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation.

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    Garriock, Robert J; Chalamalasetty, Ravindra B; Kennedy, Mark W; Canizales, Lauren C; Lewandoski, Mark; Yamaguchi, Terry P

    2015-05-01

    In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/β-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2(+)T(+) multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/β-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors.

  1. Suicide gene reveals the myocardial neovascularization role of mesenchymal stem cells overexpressing CXCR4 (MSC(CXCR4.

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    Jialiang Liang

    Full Text Available BACKGROUND: Our previous studies indicated that MSC(CXCR4 improved cardiac function after myocardial infarction (MI. This study was aimed to investigate the specific role of MSC(CXCR4 in neovascularization of infarcted myocardium using a suicide gene approach. METHODS: MSCs were transduced with either lentivirus-null vector/GFP (MSC(Null as control or vector encoding for overexpressing CXCR4/GFP. The MSC derived-endothelial cell (EC differentiation was assessed by a tube formation assay, Dil-ac-LDL uptake, EC marker expression, and VE-cadherin promoter activity assay. Gene expression was analyzed by quantitative RT-PCR or Western blot. The suicide gene approach was under the control of VE-cadherin promoter. In vivo studies: Cell patches containing MSC(Null or MSC(CXCR4 were transduced with suicide gene and implanted into the myocardium of MI rat. Rats received either ganciclovir (GCV or vehicle after cell implantation. After one month, the cardiac functional changes and neovascularization were assessed by echocardiography, histological analysis, and micro-CT imaging. RESULTS: The expression of VEGF-A and HIF-1α was significantly higher in MSC(CXCR4 as compared to MSC(Null under hypoxia. Additionally, MSC(CXCR4 enhanced new vessel formation and EC differentiation, as well as STAT3 phosphorylation under hypoxia. STAT3 participated in the transcription of VE-cadherin in MSC(CXCR4 under hypoxia, which was inhibited by WP1066 (a STAT3 inhibitor. In addition, GCV specifically induced death of ECs with suicide gene activation. In vivo studies: MSC(CXCR4 implantation promoted cardiac functional restoration, reduced infarct size, improved cardiac remodeling, and enhanced neovascularization in ischemic heart tissue. New vessels derived from MSC(CXCR4 were observed at the injured heart margins and communicated with native coronary arteries. However, the derived vessel networks were reduced by GCV, reversing improvement of cardiac function. CONCLUSION: The

  2. Functional dissection of the Oct6 schwann cell enhancer reveals an essential role for dimeric sox10 binding

    NARCIS (Netherlands)

    N.B. Jagalur (Noorjahan ); M. Ghazvini (Mehrnaz); W. Mandemakers (Wim); S. Driegen (Siska); A. Maas (Alex); E.A. Jones (Erin); F.G. Grosveld (Frank); M. Jaegle (Martine); J. Svaren (John); D.N. Meijer (Dies)

    2011-01-01

    textabstractThe POU domain transcription factor Pou3f1 (Oct6/Scip/Tst1) initiates the transition from ensheathing, promyelinating Schwann cells to myelinating cells. Axonal and other extracellular signals regulate Oct6 expression through the Oct6 Schwann cell enhancer (SCE), which is both required a

  3. Comprehensive transcriptional and functional analyses of melatonin synthesis genes in cassava reveal their novel role in hypersensitive-like cell death

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    Wei, Yunxie; Hu, Wei; Wang, Qiannan; Liu, Wei; Wu, Chunjie; Zeng, Hongqiu; Yan, Yu; Li, Xiaolin; He, Chaozu; Shi, Haitao

    2016-01-01

    Melatonin is a widely known hormone in animals. Since melatonin was discovered in plants, more and more studies highlight its involvement in a wide range of physiological processes including plant development and stress responses. Many advances have been made in the terms of melatonin-mediated abiotic stress resistance and innate immunity in plants, focusing on model plants such as rice and Arabidopsis. In this study, 7 melatonin synthesis genes were systematically analyzed in cassava. Quantitative real-time PCR showed that all these genes were commonly regulated by melatonin, flg22, Xanthomonas axonopodis pv manihotis (Xam) and hydrogen peroxide (H2O2). Transient expression in Nicotiana benthamiana revealed the subcellular locations and possible roles of these melatonin synthesis genes. Notably, we highlight novel roles of these genes in hypersensitive-like cell death, as confirmed by the results of several physiological parameters. Moreover, transient expression of these genes had significant effects on the transcripts of reactive oxygen species (ROS) accumulation and defense-related genes, and triggered the burst of callose depositions and papillae-associated plant defense, indicating the possible role of them in plant innate immunity. Taken together, this study reveals the comprehensive transcripts and putative roles of melatonin synthesis genes as well as melatonin in immune responses in cassava. PMID:27739451

  4. The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression

    NARCIS (Netherlands)

    C. Orelio; K.N. Harvey; C. Miles; R.A. Oostendorp (Robert); K. van der Horn; E.A. Dzierzak (Elaine)

    2004-01-01

    textabstractApoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in

  5. Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

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    Jarman Andrew P

    2007-05-01

    Full Text Available Abstract Background Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs, somatic gonadal precursors (SGPs, and in males, male-specific somatic gonadal precursors (msSGPs. These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. Results We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. Conclusion Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters

  6. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

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    Julie eMcLean

    2015-04-01

    Full Text Available AIMS: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM. Results: One-hundred and fifty-eight proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass.

  7. A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy

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    Sharad Kholia

    2015-06-01

    Full Text Available Introduction: Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs and has been linked to various cancers. Cellular microvesicle (MV release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression. Background: We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am. We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment. Methods: Prostate cancer cells (PC3 were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation. Conclusion: We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3 to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX treatment of

  8. In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria.

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    Henrique Borges da Silva

    2015-02-01

    Full Text Available Dendritic cells (DCs are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip, with Plasmodium chabaudi AS (Pc parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

  9. In Vivo Approaches Reveal a Key Role for DCs in CD4+ T Cell Activation and Parasite Clearance during the Acute Phase of Experimental Blood-Stage Malaria

    Science.gov (United States)

    Borges da Silva, Henrique; Fonseca, Raíssa; Cassado, Alexandra dos Anjos; Machado de Salles, Érika; de Menezes, Maria Nogueira; Langhorne, Jean; Perez, Katia Regina; Cuccovia, Iolanda Midea; Ryffel, Bernhard; Barreto, Vasco M.; Marinho, Cláudio Romero Farias; Boscardin, Silvia Beatriz; Álvarez, José Maria; D’Império-Lima, Maria Regina; Tadokoro, Carlos Eduardo

    2015-01-01

    Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection. PMID:25658925

  10. Analysis of Drosophila p8 and p52 mutants reveals distinct roles for the maintenance of TFIIH stability and male germ cell differentiation

    Science.gov (United States)

    Cruz-Becerra, Grisel; Juárez, Mandy; Valadez-Graham, Viviana

    2016-01-01

    Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.

  11. Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Fowell, Andrew J., E-mail: ajf2@soton.ac.uk [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom); Collins, Jane E.; Duncombe, Dale R.; Pickering, Judith A. [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom); Rosenberg, William M.C. [Centre for Hepatology, Division of Medicine, University College London, London (United Kingdom); Benyon, R. Christopher [Liver and Pancreas Group, University of Southampton, Division of Infection, Inflammation and Immunity, Southampton General Hospital, Southampton (United Kingdom)

    2011-04-08

    Research highlights: {yields} Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis. {yields} We used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. {yields} Specific silencing of TIMP-1, but not TIMP-2, significantly reduces HSC proliferation and is associated with reduced Akt phosphorylation. {yields} TIMP-1 is localised in part to the HSC nucleus. {yields} TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. -- Abstract: Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover

  12. Long Non-coding RNAs Expression Profile in HepG2 Cells Reveals the Potential Role of Long Non-coding RNAs in the Cholesterol Metabolism

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    Gang Liu

    2015-01-01

    Full Text Available Background: Green tea has been shown to improve cholesterol metabolism in animal studies, but the molecular mechanisms underlying this function have not been fully understood. Long non-coding RNAs (lncRNAs have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Our aim was to identify important lncRNAs that might play an important role in contributing to the benefits of epigallocatechin-3-gallate (EGCG on cholesterol metabolism. Methods: Microarrays was used to reveal the lncRNA and mRNA profiles in green tea polyphenol(--epigallocatechin gallate in cultured human liver (HepG2 hepatocytes treated with EGCG and bioinformatic analyses of the predicted target genes were performed to identify lncRNA-mRNA targeting relationships. RNA interference was used to investigate the role of lncRNAs in cholesterol metabolism. Results: The expression levels of 15 genes related to cholesterol metabolism and 285 lncRNAs were changed by EGCG treatment. Bioinformatic analysis found five matched lncRNA-mRNA pairs for five differentially expressed lncRNAs and four differentially expressed mRNA. In particular, the lncRNA AT102202 and its potential targets mRNA-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR were identified. Using a real-time polymerase chain reaction technique, we confirmed that EGCG down-regulated mRNA expression level of the HMGCR and up-regulated expression of AT102202. After AT102202 knockdown in HepG2, we observed that the level of HMGCR expression was significantly increased relative to the scrambled small interfering RNA control (P < 0.05. Conclusions: Our results indicated that EGCG improved cholesterol metabolism and meanwhile changed the lncRNAs expression profile in HepG2 cells. LncRNAs may play an important role in the cholesterol metabolism.

  13. Hydrogen Peroxide-induced Cell Death in Arabidopsis : Transcriptional and Mutant Analysis Reveals a Role of an Oxoglutarate-dependent Dioxygenase Gene in the Cell Death Process

    NARCIS (Netherlands)

    Gechev, Tsanko S.; Minkov, Ivan N.; Hille, Jacques

    2005-01-01

    Hydrogen peroxide is a major regulator of plant programmed cell death (PCD) but little is known about the downstream genes from the H2O2-signaling network that mediate the cell death. To address this question, a novel system for studying H2O2-induced programmed cell death in Arabidopsis thaliana was

  14. Long Non-coding RNAs Expression Profile in HepG2 Cells Reveals the Potential Role of Long Non-coding RNAs in the Cholesterol Metabolism

    Institute of Scientific and Technical Information of China (English)

    Gang Liu; Xinxin Zheng; Yanlu Xu; Jie Lu; Jingzhou Chen; Xiaohong Huang

    2015-01-01

    Background:Green tea has been shown to improve cholesterol metabolism in animal studies,but the molecular mechanisms underlying this function have not been fully understood.Long non-coding RNAs (lncRNAs) have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases.Our aim was to identify important lncRNAs that might play an important role in contributing to the benefits of epigallocatechin-3-gallate (EGCG) on cholesterol metabolism.Methods:Microarrays was used to reveal the lncRNA and mRNA profiles in green tea polyphenol(-)-epigallocatechin gallate in cultured human liver (HepG2) hepatocytes treated with EGCG and bioinformatic analyses of the predicted target genes were performed to identify lncRNA-mRNA targeting relationships.RNA interference was used to investigate the role of lncRNAs in cholesterol metabolism.Results:The expression levels of 15 genes related to cholesterol metabolism and 285 lncRNAs were changed by EGCG treatment.Bioinformatic analysis found five matched lncRNA-mRNA pairs for five differentially expressed lncRNAs and four differentially expressed mRNA.In particular,the lncRNA4 T102202 and its potential targets mRNA-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) were identified.Using a real-time polymerase chain reaction technique,we confirmed that EGCG down-regulated mRNA expression level of the HMGCR and up-regulated expression ofAT102202.After AT102202 knockdown in HepG2,we observed that the level of HMGCR expression was significantly increased relative to the scrambled small interfering RNA control (P < 0.05).Conclusions:Our results indicated that EGCG improved cholesterol metabolism and meanwhile changed the lncRNAs expression profile in HepG2 cells.LncRNAs may play an important role in the cholesterol metabolism.

  15. CRISPR/Cas9n-Mediated Deletion of the Snail 1Gene (SNAI1 Reveals Its Role in Regulating Cell Morphology, Cell-Cell Interactions, and Gene Expression in Ovarian Cancer (RMG-1 Cells.

    Directory of Open Access Journals (Sweden)

    Misako Haraguchi

    Full Text Available Snail1 is a transcription factor that induces the epithelial to mesenchymal transition (EMT. During EMT, epithelial cells lose their junctions, reorganize their cytoskeletons, and reprogram gene expression. Although Snail1 is a prominent repressor of E-cadherin transcription, its precise roles in each of the phenomena of EMT are not completely understood, particularly in cytoskeletal changes. Previous studies have employed gene knockdown systems to determine the functions of Snail1. However, incomplete protein knockdown is often associated with these systems, which may cause incorrect interpretation of the data. To more precisely evaluate the functions of Snail1, we generated a stable cell line with a targeted ablation of Snail1 (Snail1 KO by using the CRISPR/Cas9n system. Snail1 KO cells show increased cell-cell adhesion, decreased cell-substrate adhesion and cell migration, changes to their cytoskeletal organization that include few stress fibers and abundant cortical actin, and upregulation of epithelial marker genes such as E-cadherin, occludin, and claudin-1. However, morphological changes were induced by treatment of Snail1 KO cells with TGF-beta. Other transcription factors that induce EMT were also induced by treatment with TGF-beta. The precise deletion of Snail1 by the CRISPR/Cas9n system provides clear evidence that loss of Snail1 causes changes in the actin cytoskeleton, decreases cell-substrate adhesion, and increases cell-cell adhesion. Treatment of RMG1 cells with TGF-beta suggests redundancy among the transcription factors that induce EMT.

  16. Deletion of Slam locus in mice reveals inhibitory role of SLAM family in NK cell responses regulated by cytokines and LFA-1.

    Science.gov (United States)

    Guo, Huaijian; Cranert, Stacey A; Lu, Yan; Zhong, Ming-Chao; Zhang, Shaohua; Chen, Jun; Li, Rui; Mahl, Sarah E; Wu, Ning; Davidson, Dominique; Waggoner, Stephen N; Veillette, André

    2016-09-19

    Signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) can mediate either activating or inhibitory effects during natural killer cell (NK cell) activation. In this study, we addressed the global role, regulation, and mechanism of action of the SLAM family in NK cells by analyzing a mouse lacking the entire ∼400-kilobase Slam locus, which encodes all six SFRs and CD48, the ligand of SFR 2B4. This mouse displayed enhanced NK cell activation responses toward hematopoietic target cells. Analyses of mice lacking individual SFRs showed that the inhibitory function of the Slam locus was due solely to 2B4 and was not influenced positively or negatively by other SFRs. Differences in NK cell responses between recognition of targets expressing or lacking ligands for SFRs were enhanced by IL-12 but suppressed by type I interferon. Cytokines also changed the levels of SLAM-associated protein adaptors, which prevent the inhibitory function of SFRs. The enhanced activation responses of SFR-deficient NK cells were dependent on integrin LFA-1 but not on DNAM-1 or NKG2D. SFR-mediated inhibition prevented the generation of activated forms of LFA-1. Hence, the Slam locus has an overall inhibitory role during NK cell activation that is solely dependent on 2B4. This effect is influenced by cytokines and leads to suppression of LFA-1 activity.

  17. Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling.

    Science.gov (United States)

    Tamba, Shigero; Yodoi, Rieko; Morimoto, Kazushi; Inazumi, Tomoaki; Sukeno, Mamiko; Segi-Nishida, Eri; Okuno, Yasushi; Tsujimoto, Gozoh; Narumiya, Shuh; Sugimoto, Yukihiko

    2010-06-01

    To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.

  18. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells.

    Science.gov (United States)

    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Brandner, Johanna M

    2011-04-01

    During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non-diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non-diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non-diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non-diabetic cells. The cause of the discrepancy between diabetic and non-diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368-phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

  19. Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development.

    Science.gov (United States)

    Lin, Lin; Zhong, Si-Hui; Cui, Xiao-Feng; Li, Jianming; He, Zu-Hua

    2012-12-01

    The balance between cell proliferation and cell differentiation is essential for leaf patterning. However, identification of the factors coordinating leaf patterning and cell growth behavior is challenging. Here, we characterized a temperature-sensitive Arabidopsis mutant with leaf blade and venation defects. We mapped the mutation to the sub-2 allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) receptor-like kinase gene whose functions in leaf development have not been demonstrated. The sub-2 mutant displayed impaired blade development, asymmetric leaf shape and altered venation patterning under high ambient temperature (30°C), but these defects were less pronounced at normal growth temperature (22°C). Loss of SCM/SUB function results in reduced cell proliferation and abnormal cell expansion, as well as altered auxin patterning. SCM/SUB is initially expressed throughout leaf primordia and becomes restricted to the vascular cells, coinciding with its roles in early leaf patterning and venation formation. Furthermore, constitutive expression of the SCM/SUB gene also restricts organ growth by inhibiting the transition from cell proliferation to expansion. We propose the existence of a SCM/SUB-mediated developmental stage-specific signal for leaf patterning, and highlight the importance of the balance between cell proliferation and differentiation for leaf morphogenesis.

  20. Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance.

    Science.gov (United States)

    Kidder, Benjamin L; Palmer, Stephen

    2010-04-01

    Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs-TCFAP2C, EOMES, ETS2, and GATA3-and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell-specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.

  1. A new loss-of-function allele 28y reveals a role of ARGONAUTE1 in limiting asymmetric division of stomatal lineage ground cell

    Institute of Scientific and Technical Information of China (English)

    Kezhen Yangy; Min Jiangy; Jie Le

    2014-01-01

    In Arabidopsis thaliana L., stomata are produced through a series of divisions including asymmetric and symmetric divisions. Asymmetric entry division of meristemoid mother cellproduces two daughter cells, the smal er meristemoid and the larger sister cell, a stomatal lineage ground cell(SLGC). Stomatal lineage ground cells can differentiate into epidermal pavement cells but have the potential to divide asymmetrical y, spacing divisions, to create satel ite meristemoids. Peptide ligands and TOO MANY MOUTHS (TMM) and ERECTA family receptors regulate the initiation of stomatal lineages, activity, and orientation of spacing divisions. Here, we reported that a natural mutant 28y displayed an increased stomatal density and index. Using map-based cloning, we identified mutation in ARGONAUTE1 (AGO1) as the cause of 28y phenotypes. Time-lapse tracing of stomatal lineage cells reveals that stomatal overproduction in 28y is caused by the excessive asymmetric spacing division of SLGCs.Further genetic results demonstrated that AGO1 acts down-stream of TMM and negatively regulates the SPCH transcripts, but in a brassinosteroid-independent manner. Upregulation of AGAMOUS-LIKE16 (AGL16) in 28y mutants suggests that AGO1 is required to restrict AGL16-mediated stomatal spacing divisions, an miRNA pathway in addition to ligand-receptor signaling modules.

  2. Conditional IL-4/IL-13-deficient mice reveal a critical role of innate immune cells for protective immunity against gastrointestinal helminths.

    Science.gov (United States)

    Oeser, K; Schwartz, C; Voehringer, D

    2015-05-01

    Approximately one-third of the world population is infected with gastrointestinal helminths. Studies in mouse models have demonstrated that the cytokines interleukin (IL)-4 and IL-13 are essential for worm expulsion, but the critical cellular source of these cytokines is poorly defined. Here, we compared the immune response to Nippostrongylus brasiliensis in wild-type, T cell-specific IL-4/IL-13-deficient and general IL-4/IL-13-deficient mice. We show that T cell-derived IL-4/IL-13 promoted T helper 2 (Th2) polarization in a paracrine manner, differentiation of alternatively activated macrophages, and tissue recruitment of innate effector cells. However, innate IL-4/IL-13 played the critical role for induction of goblet cell hyperplasia and secretion of effector molecules like Mucin5ac and RELMβ in the small intestine. Surprisingly, T cell-specific IL-4/IL-13-deficient and wild-type mice cleared the parasite with comparable efficiency, whereas IL-4/IL-13-deficient mice showed impaired expulsion. These findings demonstrate that IL-4/IL-13 produced by cells of the innate immune system is required and sufficient to initiate effective type 2 immune responses resulting in protective immunity against N. brasiliensis.

  3. Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis.

    Directory of Open Access Journals (Sweden)

    Manoj B Menon

    2014-08-01

    Full Text Available Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

  4. Toward Revealing the Critical Role of Perovskite Coverage in Highly Efficient Electron-Transport Layer-Free Perovskite Solar Cells: An Energy Band and Equivalent Circuit Model Perspective.

    Science.gov (United States)

    Huang, Like; Xu, Jie; Sun, Xiaoxiang; Du, Yangyang; Cai, Hongkun; Ni, Jian; Li, Juan; Hu, Ziyang; Zhang, Jianjun

    2016-04-20

    Currently, most efficient perovskite solar cells (PVKSCs) with a p-i-n structure require simultaneously electron transport layers (ETLs) and hole transport layers (HTLs) to help collecting photogenerated electrons and holes for obtaining high performance. ETL free planar PVKSC is a relatively new and simple structured solar cell that gets rid of the complex and high temperature required ETL (such as compact and mesoporous TiO2). Here, we demonstrate the critical role of high coverage of perovskite in efficient ETL free PVKSCs from an energy band and equivalent circuit model perspective. From an electrical point of view, we confirmed that the low coverage of perovskite does cause localized short circuit of the device. With coverage optimization, a planar p-i-n(++) device with a power conversion efficiency of over 11% was achieved, implying that the ETL layer may not be necessary for an efficient device as long as the perovskite coverage is approaching 100%.

  5. Altered Macrophage and Dendritic Cell Response in Mif−/− Mice Reveals a Role of Mif for Inflammatory-Th1 Response in Type 1 Diabetes

    Science.gov (United States)

    Vazquez-Mendoza, Alicia

    2016-01-01

    Macrophage migration inhibitory factor (Mif) is highly expressed in type 1 diabetes mellitus (T1DM). However, there is limited information about how Mif influences the activation of macrophages (Mφ) and dendritic cells (DC) in T1DM. To address this issue, we induced T1DM by administering multiple low doses of streptozotocin (STZ) to Mif−/− or wild-type (Wt) BALB/c mice. We found that Mif−/− mice treated with STZ (Mif−/−STZ) developed lower levels of hyperglycemia, inflammatory cytokines, and specific pancreatic islet antigen- (PIAg-) IgG and displayed reduced cellular infiltration into the pancreatic islets compared to Wt mice treated with STZ (WtSTZ). Moreover, Mφ and DC from Mif−/−STZ displayed lower expression of MHC-II, costimulatory molecules CD80, CD86, and CD40, Toll-like receptor- (TLR-) 2, and TLR-4 than WtSTZ. These changes were associated with a reduced capacity of Mφ and DC from Mif−/−STZ to induce proliferation in ovalbumin-specific T cells. All the deficiencies observed in Mif−/−STZ were recovered by exogenous administration of recombinant Mif. These findings suggest that Mif plays a role in the molecular mechanisms of Mφ and DC activation and drives T cell responses involved in the pathology of T1DM. Therefore, Mif is a potential therapeutic target to reduce the pathology of T1DM. PMID:27699180

  6. Altered Macrophage and Dendritic Cell Response in Mif−/− Mice Reveals a Role of Mif for Inflammatory-Th1 Response in Type 1 Diabetes

    Directory of Open Access Journals (Sweden)

    Yuriko Itzel Sánchez-Zamora

    2016-01-01

    Full Text Available Macrophage migration inhibitory factor (Mif is highly expressed in type 1 diabetes mellitus (T1DM. However, there is limited information about how Mif influences the activation of macrophages (Mφ and dendritic cells (DC in T1DM. To address this issue, we induced T1DM by administering multiple low doses of streptozotocin (STZ to Mif−/− or wild-type (Wt BALB/c mice. We found that Mif−/− mice treated with STZ (Mif−/−STZ developed lower levels of hyperglycemia, inflammatory cytokines, and specific pancreatic islet antigen- (PIAg- IgG and displayed reduced cellular infiltration into the pancreatic islets compared to Wt mice treated with STZ (WtSTZ. Moreover, Mφ and DC from Mif−/−STZ displayed lower expression of MHC-II, costimulatory molecules CD80, CD86, and CD40, Toll-like receptor- (TLR- 2, and TLR-4 than WtSTZ. These changes were associated with a reduced capacity of Mφ and DC from Mif−/−STZ to induce proliferation in ovalbumin-specific T cells. All the deficiencies observed in Mif−/−STZ were recovered by exogenous administration of recombinant Mif. These findings suggest that Mif plays a role in the molecular mechanisms of Mφ and DC activation and drives T cell responses involved in the pathology of T1DM. Therefore, Mif is a potential therapeutic target to reduce the pathology of T1DM.

  7. Integrative analysis of circadian transcriptome and metabolic network reveals the role of de novo purine synthesis in circadian control of cell cycle.

    Science.gov (United States)

    Li, Ying; Li, Guang; Görling, Benjamin; Luy, Burkhard; Du, Jiulin; Yan, Jun

    2015-02-01

    Metabolism is the major output of the circadian clock in many organisms. We developed a computational method to integrate both circadian gene expression and metabolic network. Applying this method to zebrafish circadian transcriptome, we have identified large clusters of metabolic genes containing mostly genes in purine and pyrimidine metabolism in the metabolic network showing similar circadian phases. Our metabolomics analysis found that the level of inosine 5'-monophosphate (IMP), an intermediate metabolite in de novo purine synthesis, showed significant circadian oscillation in larval zebrafish. We focused on IMP dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis, with three circadian oscillating gene homologs: impdh1a, impdh1b and impdh2. Functional analysis revealed that impdh2 contributes to the daily rhythm of S phase in the cell cycle while impdh1a contributes to ocular development and pigment synthesis. The three zebrafish homologs of impdh are likely regulated by different circadian transcription factors. We propose that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is an important mechanism conferring circadian rhythmicity on the cell cycle. Our method is widely applicable to study the impact of circadian transcriptome on metabolism in complex organisms.

  8. Transcriptome-wide mapping of pea seed ageing reveals a pivotal role for genes related to oxidative stress and programmed cell death.

    Directory of Open Access Journals (Sweden)

    Hongying Chen

    Full Text Available Understanding of seed ageing, which leads to viability loss during storage, is vital for ex situ plant conservation and agriculture alike. Yet the potential for regulation at the transcriptional level has not been fully investigated. Here, we studied the relationship between seed viability, gene expression and glutathione redox status during artificial ageing of pea (Pisum sativum seeds. Transcriptome-wide analysis using microarrays was complemented with qRT-PCR analysis of selected genes and a multilevel analysis of the antioxidant glutathione. Partial degradation of DNA and RNA occurred from the onset of artificial ageing at 60% RH and 50°C, and transcriptome profiling showed that the expression of genes associated with programmed cell death, oxidative stress and protein ubiquitination were altered prior to any sign of viability loss. After 25 days of ageing viability started to decline in conjunction with progressively oxidising cellular conditions, as indicated by a shift of the glutathione redox state towards more positive values (>-190 mV. The unravelling of the molecular basis of seed ageing revealed that transcriptome reprogramming is a key component of the ageing process, which influences the progression of programmed cell death and decline in antioxidant capacity that ultimately lead to seed viability loss.

  9. An ENU-induced mouse mutant of SHIP1 reveals a critical role of the stem cell isoform for suppression of macrophage activation.

    Science.gov (United States)

    Nguyen, Nhu-Y N; Maxwell, Mhairi J; Ooms, Lisa M; Davies, Elizabeth M; Hilton, Adrienne A; Collinge, Janelle E; Hilton, Douglas J; Kile, Benjamin T; Mitchell, Christina A; Hibbs, Margaret L; Jane, Stephen M; Curtis, David J

    2011-05-19

    In a recessive ENU mutagenesis screen for embryonic lethality, we identified a mouse pedigree with a missense mutation of SHIP1 (SHIP1(el20)) leading to an amino acid substitution I641T in the inositol-5'-phosphatase domain that represses phosphatidylinositol-3-kinase signaling. Despite detectable expression of functional SHIP1 protein, the phenotype of homozygous SHIP1(el20/el20) mice was more severe than gene-targeted SHIP1-null (SHIP1(-/-)) mice. Compared with age-matched SHIP1(-/-) mice, 5-week-old SHIP1(el20/el20) mice had increased myeloid cells, serum IL-6 levels, marked reductions in lymphoid cells, and died by 7 weeks of age with infiltration of the lungs by activated macrophages. Bone marrow transplantation demonstrated that these defects were hematopoietic-cell-autonomous. We show that the el20 mutation reduces expression in SHIP1(el20/el20) macrophages of both SHIP1 and s-SHIP, an isoform of SHIP1 generated by an internal promoter. In contrast, SHIP1(-/-) macrophages express normal levels of s-SHIP. Compound heterozygous mice (SHIP1(-/el20)) had the same phenotype as SHIP1(-/-) mice, thus providing genetic proof that the more severe phenotype of SHIP1(el20/el20) mice is probably the result of concomitant loss of SHIP1 and s-SHIP. Our results suggest that s-SHIP synergizes with SHIP1 for suppression of macrophage activation, thus providing the first evidence for a role of s-SHIP in adult hematopoiesis.

  10. Integrated network analysis reveals a novel role for the cell cycle in 2009 pandemic influenza virus-induced inflammation in macaque lungs

    Directory of Open Access Journals (Sweden)

    Shoemaker Jason E

    2012-08-01

    Full Text Available Abstract Background Annually, influenza A viruses circulate the world causing wide-spread sickness, economic loss, and death. One way to better defend against influenza virus-induced disease may be to develop novel host-based therapies, targeted at mitigating viral pathogenesis through the management of virus-dysregulated host functions. However, mechanisms that govern aberrant host responses to influenza virus infection remain incompletely understood. We previously showed that the pandemic H1N1 virus influenza A/California/04/2009 (H1N1; CA04 has enhanced pathogenicity in the lungs of cynomolgus macaques relative to a seasonal influenza virus isolate (A/Kawasaki/UTK-4/2009 (H1N1; KUTK4. Results Here, we used microarrays to identify host gene sequences that were highly differentially expressed (DE in CA04-infected macaque lungs, and we employed a novel strategy – combining functional and pathway enrichment analyses, transcription factor binding site enrichment analysis and protein-protein interaction data – to create a CA04 differentially regulated host response network. This network describes enhanced viral RNA sensing, immune cell signaling and cell cycle arrest in CA04-infected lungs, and highlights a novel, putative role for the MYC-associated zinc finger (MAZ transcription factor in regulating these processes. Conclusions Our findings suggest that the enhanced pathology is the result of a prolonged immune response, despite successful virus clearance. Most interesting, we identify a mechanism which normally suppresses immune cell signaling and inflammation is ineffective in the pH1N1 virus infection; a dyregulatory event also associated with arthritis. This dysregulation offers several opportunities for developing strain-independent, immunomodulatory therapies to protect against future pandemics.

  11. Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Hironori Waki

    2011-10-01

    Full Text Available Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type-specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq. FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA-mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our

  12. A suppressor/enhancer screen in Drosophila reveals a role for wnt-mediated lipid metabolism in primordial germ cell migration.

    Directory of Open Access Journals (Sweden)

    Mark A McElwain

    Full Text Available Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P, the product of Dcerk, allowing proper PGC migration.

  13. High-Throughput Flow Cytometry Screening Reveals a Role for Junctional Adhesion Molecule A as a Cancer Stem Cell Maintenance Factor

    DEFF Research Database (Denmark)

    Lathia, Justin D; Li, Meizhang; Sinyuk, Maksim

    2014-01-01

    Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow...

  14. Three-dimensional culture of sebaceous gland cells revealing the role of prostaglandin E{sub 2}-induced activation of canonical Wnt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Go J., E-mail: medical21go@yahoo.co.jp; Saya, Hideyuki

    2013-09-06

    Highlights: •Three-dimensional culture generates “semi-vivo” sebaceous glands. •Xenograft model failed to mimic the biology of sebaceous glands in vivo. •Proinflammatory cytokine PGE{sub 2} enhances Wnt signal activity in the organoids. •PGE{sub 2} influences on the mitochondrial and lipid metabolism in the organoids. •Considering 3R agenda, “semi-vivo” sebaceous glands are useful for research. -- Abstract: Background: Prostaglandin E{sub 2} (PGE{sub 2}) is a proinflammatory mediator and activates the canonical Wnt–β-catenin signaling pathway in hematopoietic stem cells. The SZ95 cell line was established from human sebaceous gland cells and is studied as a model system for these cells. Given that 2D culture of SZ95 cells does not recapitulate the organization of sebaceous glands in situ, we developed a 3D culture system for these cells and examined the effects of PGE{sub 2} on cell morphology and function. Results: SZ95 cells maintained in 3D culture formed organoids that mimicked the organization of sebaceous glands in situ, including the establishment of a basement membrane. Organoids exposed to PGE{sub 2} were larger and adopted a more complex organization compared with control organoids. PGE{sub 2} activated the canonical Wnt signaling pathway as well as increased cell viability and proliferation, mitochondrial metabolism, and lipid synthesis in the organoids. Conclusions: Culture of SZ95 cells in 3D culture system recapitulates the structure and susceptibility to PGE{sub 2} of sebaceous glands in situ and should prove useful for studies of the response of these glands to inflammation and other environmental stressors. Our results also implicate PGE{sub 2}-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of “semi-vivo” sebaceous glands.

  15. Assessment of CD4+ T cell responses to glutamic acid decarboxylase 65 using DQ8 tetramers reveals a pathogenic role of GAD65 121-140 and GAD65 250-266 in T1D development.

    Directory of Open Access Journals (Sweden)

    I-Ting Chow

    Full Text Available Susceptibility to type 1 diabetes (T1D is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02. Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65 using DQ8 tetramers. We demonstrated that GAD65 121-140 and GAD65 250-266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65 121-140 and GAD65 250-266 carried a Th1-dominant phenotype, with some of the GAD65 121-140-specific T cell clones producing IL-17. GAD65 250-266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs revealed that GAD65 250-266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65 121-140 and GAD65 250-266 epitopes and implicate their possible contribution to the progression of T1D.

  16. Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

    Directory of Open Access Journals (Sweden)

    Aristidis Kritis

    Full Text Available Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD or ischemic preconditioning (IPS. Nerve Growth Factor (NGF treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2/CO(2 (93:5% in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

  17. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells

    OpenAIRE

    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Johanna M Brandner

    2010-01-01

    Abstract During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its t...

  18. Suppressor screen and phenotype analyses revealed an emerging role of the Monofunctional peroxisomal enoyl-CoA hydratase 2 in compensated cell enlargement

    Directory of Open Access Journals (Sweden)

    Mana eKatano

    2016-02-01

    Full Text Available Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves β-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H+-PPase activity is required for gluconeogenesis. Lack of H+-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE. Here, we mutagenized fugu5-1 seeds with 12C6+ heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm, similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2 as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1 and A#3-1;fugu5-1. Taken together, these results suggest that defects in either H+-PPase or ECH2 compromise cell proliferation due to defects in mobilizing stored lipids. In contrast, ECH2 alone likely promotes CCE during the post-mitotic cell

  19. Transcriptional profiling of human breast cancer cells cultured under microgravity conditions revealed the key role of genetic gravity sensors previously detected in Drosophila melanogaster

    Science.gov (United States)

    Valdivia-Silva, Julio E.; Lavan, David; Diego Orihuela-Tacuri, M.; Sanabria, Gabriela

    2016-07-01

    Currently, studies in Drosophila melanogaster has shown emerging evidence that microgravity stimuli can be detected at the genetic level. Analysis of the transcriptome in the pupal stage of the fruit flies under microgravity conditions versus ground controls has suggested the presence of a few candidate genes as "gravity sensors" which are experimentally validated. Additionally, several studies have shown that microgravity causes inhibitory effects in different types of cancer cells, although the genes involved and responsible for these effects are still unknown. Here, we demonstrate that the genes suggested as the sensors of gravitational waves in Drosophila melanogaster and their human counterpart (orthologous genes) are highly involved in carcinogenesis, proliferation, anti-apoptotic signals, invasiveness, and metastatic potential of breast cancer cell tumors. The transcriptome analyses suggested that the observed inhibitory effect in cancer cells could be due to changes in the genetic expression of these candidates. These results encourage the possibility of new therapeutic targets managed together and not in isolation.

  20. Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8¿ T cells in early viral evolution

    DEFF Research Database (Denmark)

    Callendret, Benoît; Bukh, Jens; Eccleston, Heather B;

    2011-01-01

    The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8(+) T cells is a dominant force driving early amino acid...... occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8(+) T cells. This influence of immune pressure on viral evolution appears to subside as chronic...... replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8(+) T cell responses combined with sequencing of recovered...

  1. Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

    OpenAIRE

    Yang, Yun-Gui; Saidi, Amal; Frappart, Pierre-Olivier; Min, WooKee; Barrucand, Christelle; Dumon-Jones, Valérie; Michelon, Jocelyne; Herceg, Zdenko; Wang, Zhao-Qi

    2006-01-01

    NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting...

  2. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

    NARCIS (Netherlands)

    Boerboom, A.M.J.F.; Vermeulen, M.; Woude, van der H.; Bremer, B.I.; Lee, Y.Y.; Kampman, E.; Bladeren, van P.J.; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.

    2006-01-01

    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  3. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

    NARCIS (Netherlands)

    Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.

    2006-01-01

    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  4. Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization

    NARCIS (Netherlands)

    Denholtz, M.; Bonora, G.; Chronis, C.; Splinter, E.; de Laat, W.; Ernst, J.; Pellegrini, M.; Plath, K.

    2013-01-01

    The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gra

  5. Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans

    Directory of Open Access Journals (Sweden)

    Gao Qingshen

    2007-01-01

    Full Text Available Abstract Background The four highly homologous human EHD proteins (EHD1-4 form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. Results Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4 but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006 Mol. Biol. Cell 17, 163, regulates transport from the early endosome to the recycling endosome. Conclusion Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.

  6. Targeting Polycomb to Pericentric Heterochromatin in Embryonic Stem Cells Reveals a Role for H2AK119u1 in PRC2 Recruitment

    Directory of Open Access Journals (Sweden)

    Sarah Cooper

    2014-06-01

    Full Text Available The mechanisms by which the major Polycomb group (PcG complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, we demonstrate that, in methylation-deficient embryonic stem cells (ESCs, CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, we find that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. We discuss our data in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.

  7. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

    DEFF Research Database (Denmark)

    Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene

    2011-01-01

    in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...... enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important......Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme...

  8. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    Science.gov (United States)

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  9. Synthetic protein interactions reveal a functional map of the cell

    Science.gov (United States)

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  10. Offenders' crime narratives as revealed by the Narrative Roles Questionnaire.

    Science.gov (United States)

    Youngs, Donna; Canter, David V

    2013-03-01

    The study of narrative processes as part of the immediate factors that shape criminal action is limited by the lack of a methodology for differentiating the narrative themes that characterise specific crime events. The current study explores how the roles offenders see themselves as playing during an offence encapsulate their underlying crime narratives and thus provide the basis for a quantitative methodology. To test this possibility, a 33-item Narrative Roles Questionnaire (NRQ) was developed from intensive interviews with offenders about their experience of committing a recent offence. A multidimensional analysis of the NRQ completed by 71 convicted offenders revealed life narrative themes similar to those identified in fiction by Frye and with noncriminals by McAdams, labelled The Professional, Victim, Hero, and Revenger offence roles. The NRQ thus is a first step in opening up the possibility of empirical studies of the narrative aetiological perspective in criminology.

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

    CERN Document Server

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

    2012-01-01

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

  12. A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

    Science.gov (United States)

    Tilgner, K; Neganova, I; Moreno-Gimeno, I; Al-Aama, J Y; Burks, D; Yung, S; Singhapol, C; Saretzki, G; Evans, J; Gorbunova, V; Gennery, A; Przyborski, S; Stojkovic, M; Armstrong, L; Jeggo, P; Lako, M

    2013-08-01

    DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

  13. Transcriptome Profiling of the C. elegans Rb Ortholog Reveals Diverse Developmental Roles

    OpenAIRE

    Kirienko, Natalia V; Fay, David S.

    2007-01-01

    LIN-35 is the single C. elegans ortholog of the mammalian pocket protein family members, pRb, p107, and p130. To gain insight into the roles of pocket proteins during development, a microarray analysis was performed with lin-35 mutants. Stage-specific regulation patterns were revealed, indicating that LIN-35 plays diverse roles at distinct developmental stages. LIN-35 was found to repress the expression of many genes involved in cell proliferation in larvae, an activity that is carried out in...

  14. Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

    Science.gov (United States)

    Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

    2015-07-01

    Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

  15. Bone marrow chimeric mice reveal a dual role for CD36 in Plasmodium berghei ANKA infection

    Directory of Open Access Journals (Sweden)

    Febbraio Maria

    2007-03-01

    Full Text Available Abstract Background Adhesion of Plasmodium-infected red blood cells (iRBC to different host cells, ranging from endothelial to red blood cells, is associated to malaria pathology. In vitro studies have shown the relevance of CD36 for adhesion phenotypes of Plasmodium falciparum iRBC such as sequestration, platelet mediated clumping and non-opsonic uptake of iRBC. Different adhesion phenotypes involve different host cells and are associated with different pathological outcomes of disease. Studies with different human populations with CD36 polymorphisms failed to attribute a clear role to CD36 expression in human malaria. Up to the present, no in vivo model has been available to study the relevance of different CD36 adhesion phenotypes to the pathological course of Plasmodium infection. Methods Using CD36-deficient mice and their control littermates, CD36 bone marrow chimeric mice, expressing CD36 exclusively in haematopoietic cells or in non-haematopoietic cells, were generated. Irradiated CD36-/- and wild type mice were also reconstituted with syngeneic cells to control for the effects of irradiation. The reconstituted mice were infected with Plasmodium berghei ANKA and analysed for the development of blood parasitaemia and neurological symptoms. Results All mice reconstituted with syngeneic bone marrow cells as well as chimeric mice expressing CD36 exclusively in non-haematopoietic cells died from experimental cerebral malaria between day 6 and 12 after infection. A significant proportion of chimeric mice expressing CD36 only in haematopoietic cells did not die from cerebral malaria. Conclusion The analysis of bone marrow chimeric mice reveals a dual role of CD36 in P. berghei ANKA infection. Expression of CD36 in haematopoietic cells, most likely macrophages and dendritic cells, has a beneficial effect that is masked in normal mice by adverse effects of CD36 expression in non-haematopoietic cells, most likely endothelial cells.

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

    Directory of Open Access Journals (Sweden)

    Anne L Fletcher

    2011-09-01

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

  17. Morphogenetic and histogenetic roles of the temporal-spatial organization of cell proliferation in the vertebrate corticogenesis as revealed by inter-specific analyses of the optic tectum cortex development

    Directory of Open Access Journals (Sweden)

    Melina eRapacioli

    2016-03-01

    Full Text Available The central nervous system areas displaying the highest structural and functional complexity correspond to the so called cortices, i.e. concentric alternating neuronal and fibrous layers. Corticogenesis, i.e. the development of the cortical organization, depends on the temporal-spatial organization of several developmental events: (a the duration of the proliferative phase of the neuroepithelium, (b the relative duration of symmetric (expansive versus asymmetric (neuronogenic sub phases, (c the spatial organization of each kind of cell division, (e the time of determination and cell cycle exit and (f the time of onset of the postmitotic neuronal migration and (g the time of onset of the neuronal structural and functional differentiation. The first five events depend on molecular mechanisms that perform a fine tuning of the proliferative activity. Changes in any of them significantly influence the cortical size or volume (tangential expansion and radial thickness, morphology, architecture and also impact on neuritogenesis and synaptogenesis affecting the cortical wiring. This paper integrates information, obtained in several species, on the developmental roles of cell proliferation in the development of the optic tectum cortex, a multilayered associative area of the dorsal (alar midbrain. The present review (1 compiles relevant information on the temporal and spatial organization of cell proliferation in different species (fish, amphibians, birds and mammals, (2 revises the main molecular events involved in the isthmic organizer determination and localization, (3 describes how the patterning installed by isthmic organizer is translated into spatially organized neural stem cell proliferation (i.e. by means of growth factors, receptors, transcription factors, signaling pathways, etc. and (4 describes the morpho- and histogenetic effect of a spatially organized cell proliferation in the above mentioned species. A brief section on the optic tectum

  18. Adult mouse cortical cell taxonomy revealed by single cell transcriptomics.

    Science.gov (United States)

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T; Sorensen, Staci A; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties.

  19. MicroRNA Profiling of the Effect of the Heptapeptide Angiotensin-(1-7) in A549 Lung Tumor Cells Reveals a Role for miRNA149-3p in Cellular Migration Processes

    Science.gov (United States)

    da Silva, Brenda de Oliveira; Lima, Kelvin Furtado; Gonçalves, Letícia Rocha; da Silveira, Marina Bonfogo; Moraes, Karen C. M.

    2016-01-01

    Lung cancer is one of the most frequent types of cancer in humans and a leading cause of death worldwide. The high mortality rates are correlated with late diagnosis, which leads to high rates of metastasis found in patients. Thus, despite all the improvement in therapeutic approaches, the development of new drugs that control cancer cell migration and metastasis are required. The heptapeptide angiotensin-(1–7) [ang-(1–7)] has demonstrated the ability to control the growth rates of human lung cancer cells in vitro and in vivo, and the elucidation of central elements that control the fine-tuning of cancer cells migration in the presence of the ang-(1–7), will support the development of new therapeutic approaches. Ang-(1–7) is a peptide hormone of the renin-angiotensin system (RAS) and this study investigates the modulatory effect of the heptapeptide on the expression pattern of microRNAs (miRNAs) in lung tumor cells, to elucidate mechanistic concerns about the effect of the peptide in the control of tumor migratory processes. Our primary aim was to compare the miRNA profiling between treated and untreated-heptapeptide cells to characterize the relevant molecule that modulates cellular migration rates. The analyses selected twenty one miRNAs, which are differentially expressed between the groups; however, statistical analyses indicated miRNA-149-3p as a relevant molecule. Once functional analyses were performed, we demonstrated that miRNA-149-3p plays a role in the cellular migration processes. This information could be useful for future investigations on drug development. PMID:27598578

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  1. A quorum-sensing factor in vegetative Dictyostelium discoideum cells revealed by quantitative migration analysis.

    Directory of Open Access Journals (Sweden)

    Laurent Golé

    Full Text Available BACKGROUND: Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS. In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. METHODS AND FINDINGS: To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (t ≤ 5 min, but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. CONCLUSION: This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase.

  2. Study of nsLTPs in Lotus japonicus genome reveal a specific epidermal cell member (LjLTP10) regulated by drought stress in aerial organs with a putative role in cutin formation.

    Science.gov (United States)

    Tapia, G; Morales-Quintana, L; Parra, C; Berbel, A; Alcorta, M

    2013-07-01

    The cuticle is the first defense against pathogens and the second way water is lost in plants. Hydrophobic layers covering aerial plant organs from primary stages of development form cuticle, including major classes of aliphatic wax components and cutin. Extensive research has been conducted to understand cuticle formation mechanisms in plants. However, many questions remain unresolved in the transport of lipid components to form cuticle. Database studies of the Lotus japonicus genome have revealed the presence of 24 sequences classified as putative non-specific lipid transfer proteins (nsLTPs), which were classified in seven groups; four groups were selected because of their expression in aerial organs. LjLTP8 forms a cluster with DIR1 in Arabidopsis thaliana while LjLTP6, LjLTP9, and LjLTP10 were grouped as type I LTPs. In silico studies showed a high level of structural conservation, and substrate affinity studies revealed palmitoyl-CoA as the most likely ligand for these LTPs, although the Lyso-Myristoyl Phosphatidyl Choline, Lyso-myristoyl phosphatidyl glycerol, and Lyso-stearyl phosphatidyl choline ligands also showed a high affinity with the proteins. The LjLTP6 and LjLTP10 genes were expressed in both the stems and the leaves under normal conditions and were highly induced during drought stress. LjLTP10 was the most induced gene in shoots during drought. The gene was only expressed in the epidermal cells of stems, primordial leaves, and young leaflets. LjLTP10 was positively regulated by MeJA but repressed by abscisic acid (ABA), ethylene, and H2O2, while LjLTP6 was weakly induced by MeJA, repressed by H2O2, and not affected by ABA and ethylene. We suggest that LjLTP10 is involved in plant development of stem and leaf cuticle, but also in acclimation to tolerate drought stress in L. japonicus.

  3. Genome-wide analyses reveal a role for peptide hormones in planarian germline development.

    Directory of Open Access Journals (Sweden)

    James J Collins

    Full Text Available Bioactive peptides (i.e., neuropeptides or peptide hormones represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.

  4. Researchers Reveal Ecological Roles of Biological Soil Crusts in Desert

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ Biological soil crust is a complex organic integrity of cyanobacteria, green algae, lichens and mosses, fungi, and other bacteria. This is a common and widespread phenomenon in desert areas all over the world. Biologically,this kind of soil crust differs a lot from physical ones in terms of physical and chemical properties, and become important biological factors in vegetation succession. Despite its unassuming appearance, the crust plays a significant role in the desert ecosystem, involving the process of soil formation, stability and fertility,the prevention of soil erosion by water or wind, the increased possibility of vascular plants colonization, and the stabilization of sand dunes.

  5. A possible role for neutrophils in allergic rhinitis revealed after cellular subclassification

    Science.gov (United States)

    Arebro, Julia; Ekstedt, Sandra; Hjalmarsson, Eric; Winqvist, Ola; Kumlien Georén, Susanna; Cardell, Lars-Olaf

    2017-01-01

    A re-examination of former concepts is required to meet today’s medical challenges in allergic rhinitis. Previously, neutrophils have been treated as a relatively homogenous cell population found in the nose both when the patient is suffering at the height of the allergic season as well as when the patient report no symptoms. However, new data indicates that neutrophils can be divided into different subsets with diverse roles in inflammation. We showed increased levels of neutrophils in peripheral blood, nasal biopsies and nasal lavage fluid (NAL) from allergic patients during the pollen season compared to healthy controls. A closer examination revealed that the activated subset of neutrophils, CD16high CD62Ldim, outweighed the normal form CD16high CD62Lhigh in nasal tissue among these patients. This skewed distribution was not seen in controls. The normal subset prevailed in peripheral blood from patients as well as controls, whereas CD16high CD62Ldim and CD16dim CD62Ldim subsets, the latter considered “end state” neutrophils before apoptosis, were elevated in NAL. Functional in vitro experiments revealed that activated neutrophils exhibit a T cell priming capacity and an ability to enhance eosinophil migration. Activated neutrophils may thus contribute to allergic inflammation seen in allergic rhinitis by priming T cells and attracting eosinophils. PMID:28272395

  6. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    Science.gov (United States)

    Laura, Richard P; Dong, David; Reynolds, Wanda F; Maki, Richard A

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

  7. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation.

    Directory of Open Access Journals (Sweden)

    Richard P Laura

    Full Text Available Among the human heme-peroxidase family, myeloperoxidase (MPO has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO after it exits the endoplasmic reticulum (ER. Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO's single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO's unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.

  8. T47D Cells Expressing Myeloperoxidase Are Able to Process, Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

    Science.gov (United States)

    Laura, Richard P.; Dong, David; Reynolds, Wanda F.; Maki, Richard A.

    2016-01-01

    Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO. PMID:26890638

  9. Role of Mesenchymal Stem Cells In Tumorigenesis

    Science.gov (United States)

    2009-08-01

    stem cells ( BMDC ), which then acts in a paracrine fashion on the cancer cells to enhance their invasion [7]. Interestingly the group of Karnoub showed...AD_________________ AWARD NUMBER: W81XWH-08-1-0523 TITLE: Role of Mesenchymal Stem Cells in...DATES COVERED 1 Aug 2008 – 31 Jul 2009 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Role of Mesenchymal Stem Cells in Tumorigenesis 5b. GRANT

  10. Conditional Knockout in Mice Reveals the Critical Roles of Ppp2ca in Epidermis Development

    Directory of Open Access Journals (Sweden)

    Chao Fang

    2016-05-01

    Full Text Available The epidermis is an important tissue in Homo sapines and other animals, and an abnormal epidermis will cause many diseases. Phosphatase 2A (PP2A is an important serine and threonine phosphatase. The α isoform of the PP2A catalytic subunit (Ppp2ca gene encoding PP2Acα is critical for cell proliferation, growth, metabolism and tumorigenesis. However, to date, no study has revealed its roles in epidermis development. To specifically investigate the roles of PP2Acα in epidermis development, we first generated Ppp2caflox/flox transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2caflox/flox; Krt14-Cre mice had significant hair loss. In addition, histological analyses showed that the morphogenesis and hair regeneration cycle of hair follicles were disrupted in these mice. Moreover, Ppp2caflox/flox; Krt14-Cre mice had smaller size, melanin deposition and hyperproliferation at the base of the claws. Accordingly, our study demonstrates that PP2Acα plays important roles in both hair follicle and epidermis development. Additionally, the Ppp2caflox/flox mice generated in this study can serve as a useful transgene model to study the roles of PP2Acα in other developmental processes and diseases.

  11. Lupus nephritis: the central role of nucleosomes revealed.

    Science.gov (United States)

    Mortensen, Elin S; Fenton, Kristin A; Rekvig, Ole P

    2008-02-01

    Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger's danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11-13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045) and Medzhitov's and Janeway's (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298-300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185-188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20

  12. Manipulation of the Gut Microbiota Reveals Role in Colon Tumorigenesis

    Science.gov (United States)

    Zackular, Joseph P.; Baxter, Nielson T.

    2015-01-01

    ABSTRACT There is growing evidence that individuals with colonic adenomas and carcinomas harbor a distinct microbiota. Alterations to the gut microbiota may allow the outgrowth of bacterial populations that induce genomic mutations or exacerbate tumor-promoting inflammation. In addition, it is likely that the loss of key bacterial populations may result in the loss of protective functions that are normally provided by the microbiota. We explored the role of the gut microbiota in colon tumorigenesis by using an inflammation-based murine model. We observed that perturbing the microbiota with different combinations of antibiotics reduced the number of tumors at the end of the model. Using the random forest machine learning algorithm, we successfully modeled the number of tumors that developed over the course of the model on the basis of the initial composition of the microbiota. The timing of antibiotic treatment was an important determinant of tumor outcome, as colon tumorigenesis was arrested by the use of antibiotics during the early inflammation period of the murine model. Together, these results indicate that it is possible to predict colon tumorigenesis on the basis of the composition of the microbiota and that altering the gut microbiota can alter the course of tumorigenesis. IMPORTANCE Mounting evidence indicates that alterations to the gut microbiota, the complex community of bacteria that inhabits the gastrointestinal tract, are strongly associated with the development of colorectal cancer. We used antibiotic perturbations to a murine model of inflammation-driven colon cancer to generate eight starting communities that resulted in various severities of tumorigenesis. Furthermore, we were able to quantitatively predict the final number of tumors on the basis of the initial composition of the gut microbiota. These results further bolster the evidence that the gut microbiota is involved in mediating the development of colorectal cancer. As a final proof of

  13. Mechanisms of cell cycle control revealed by a systematic and quantitative overexpression screen in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Wei Niu

    2008-07-01

    Full Text Available Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%-94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression.

  14. Transcriptional analysis of the endothelial response to diabetes reveals a role for galectin-3.

    Science.gov (United States)

    Darrow, April L; Shohet, Ralph V; Maresh, J Gregory

    2011-10-20

    To characterize the endothelial dysfunction associated with Type II diabetes, we surveyed transcriptional responses in the vascular endothelia of mice receiving a diabetogenic, high-fat diet. Tie2-GFP mice were fed a diet containing 60% fat calories (HFD); controls were littermates fed normal chow. Following 4, 6, and 8 wk, aortic and leg muscle tissues were enzymatically dispersed, and endothelial cells were obtained by fluorescence-activated cell sorting. Relative mRNA abundance in HFD vs. control endothelia was measured with long-oligo microarrays; highly dysregulated genes were confirmed by real-time PCR and protein quantification. HFD mice were hyperglycemic by 2 wk and displayed vascular insulin resistance and decreased glucose tolerance by 5 and 6 wk, respectively. Endothelial transcripts upregulated by HFD included galectin-3 (Lgals3), 5-lipoxygenase-activating protein, and chemokine ligands 8 and 9. Increased LGALS3 protein was detected in muscle endothelium by immunohistology accompanied by elevated LGALS3 in the serum of HFD mice. Our comprehensive analysis of the endothelial transcriptional response in a model of Type II diabetes reveals novel regulation of transcripts with roles in inflammation, insulin sensitivity, oxidative stress, and atherosclerosis. Increased endothelial expression and elevated humoral levels of LGALS3 supports a role for this molecule in the vascular response to diabetes, and its potential as a direct biomarker for the inflammatory state in diabetes.

  15. Investigating the role of retinal Müller cells with approaches in genetics and cell biology.

    Science.gov (United States)

    Fu, Suhua; Zhu, Meili; Ash, John D; Wang, Yunchang; Le, Yun-Zheng

    2014-01-01

    Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology.

  16. The role of dendritic cells in cancer

    DEFF Research Database (Denmark)

    Hansen, Morten; Andersen, Mads Hald

    2017-01-01

    Though present in low numbers, dendritic cells (DCs) are recognized as major players in the control of cancer by adaptive immunity. The roles of cytotoxic CD8+ T-cells and Th1 helper CD4+ T-cells are well-documented in murine models of cancer and associated with a profound prognostic impact when...... treatment regimens against cancer....

  17. The role of Cbln1 on Purkinje cell synapse formation.

    Science.gov (United States)

    Ito-Ishida, Aya; Okabe, Shigeo; Yuzaki, Michisuke

    2014-06-01

    Cbln1 is a glycoprotein which belongs to the C1q family. In the cerebellum, Cbln1 is produced and secreted from granule cells and works as a strong synapse organizer between Purkinje cells and parallel fibers, the axons of the granule cells. In this update article, we will describe the molecular mechanisms by which Cbln1 induces synapse formation and will review our findings on the axonal structural changes which occur specifically during this process. We will also describe our recent finding that Cbln1 has a suppressive role in inhibitory synapse formation between Purkinje cells and molecular layer interneurons. Our results have revealed that Cbln1 plays an essential role to establish parallel fiber-Purkinje cell synapses and to regulate balance between excitatory and inhibitory input on Purkinje cells.

  18. IL-15 deficient tax mice reveal a role for IL-1α in tumor immunity.

    Directory of Open Access Journals (Sweden)

    Daniel A Rauch

    Full Text Available IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/- TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.

  19. IL-15 deficient tax mice reveal a role for IL-1α in tumor immunity.

    Science.gov (United States)

    Rauch, Daniel A; Harding, John C; Ratner, Lee

    2014-01-01

    IL-15 is recognized as a promising candidate for tumor immunotherapy and has been described as both a promoter of cancer and a promoter of anti-cancer immunity. IL-15 was discovered in cells transformed by HTLV-1, the etiologic agent of adult T cell leukemia/lymphoma (ATL) and the human retrovirus that carries the Tax oncogene. We have developed the TAX-LUC mouse model of ATL in which Tax expression drives both malignant transformation and luciferase expression, enabling non-invasive imaging of tumorigenesis in real time. To identify the role of IL-15 in spontaneous development of lymphoma in vivo, an IL-15(-/-) TAX-LUC strain was developed and examined. The absence of IL-15 resulted in aggressive tumor growth and accelerated mortality and demonstrated that IL-15 was not required for Tax-mediated lymphoma but was essential for anti-tumor immunity. Further analysis revealed a unique transcriptional profile in tumor cells that arise in the absence of IL-15 that included a significant increase in the expression of IL-1α and IL-1α-regulated cytokines. Moreover, anti-IL-1α antibodies and an IL-1 receptor antagonist (Anakinra) were used to interrogate the potential of IL-1α targeted therapies in this model. Taken together, these findings identify IL-15 and IL-1α as therapeutic targets in lymphoma.

  20. Role of human mast cells and basophils in bronchial asthma.

    Science.gov (United States)

    Marone, Gianni; Triggiani, Massimo; Genovese, Arturo; De Paulis, Amato

    2005-01-01

    Mast cells and basophils are the only cells expressing the tetrameric (alphabetagamma2) structure of the high affinity receptor for IgE (FcepsilonRI) and synthesizing histamine in humans. Human FcepsilonRI+ cells are conventionally considered primary effector cells of bronchial asthma. There is now compelling evidence that these cells differ immunologically, biochemically, and pharmacologically, which suggests that they might play distinct roles in the appearance and fluctuation of the asthma phenotype. Recent data have revealed the complexity of the involvement of human mast cells and basophils in asthma and have shed light on the control of recruitment and activation of these cells in different lung compartments. Preliminary evidence suggests that these cells might not always be detrimental in asthma but, under some circumstances, they might exert a protective effect by modulating certain aspects of innate and acquired immunity and allergic inflammation.

  1. A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress.

    Directory of Open Access Journals (Sweden)

    Josh R Beck

    2013-02-01

    Full Text Available Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic

  2. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

    Directory of Open Access Journals (Sweden)

    Barbara Witek

    Full Text Available Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

  3. Revealing the role of organic cations in hybrid halide perovskite CH3NH3PbI3

    OpenAIRE

    Sanvito, Stefano; Motta, Carlo

    2015-01-01

    The hybrid halide perovskite CH$_{3}$NH$_{3}$PbI$_{3}$ has enabled solar cells to reach an efficiency of about 18\\%, demonstrating a pace for improvements with no precedents in the solar energy arena. Despite such explosive progress, the microscopic origin behind the success of such material is still debated, with the role played by the organic cations in the light-harvesting process remaining unclear. Here van-der-Waals-corrected density functional theory calculations reveal that the orienta...

  4. Organising cells into tissues: new roles for cell adhesion molecules in planar cell polarity.

    Science.gov (United States)

    Saburi, Sakura; McNeill, Helen

    2005-10-01

    Planar cell polarity (PCP) is the coordinated organization of cells within the plane of the epithelium, first described in Drosophila. A Frizzled signalling pathway dedicated to PCP (the non-canonical Frizzled pathway) acts through Dishevelled and small G proteins, as does the classical Wnt pathway, but then diverges downstream of Dishevelled. Recent studies have demonstrated a crucial role for several atypical cadherin molecules (Fat, Dachsous and Flamingo) in controlling PCP signalling. Recent work has also indicated that the first sign of PCP during development is the polarized localization of PCP proteins (Frizzled, Flamingo, Dishevelled, etc). Exciting new data reveal that this PCP pathway is conserved to man.

  5. The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.

    Science.gov (United States)

    Björklund, Åsa K; Forkel, Marianne; Picelli, Simone; Konya, Viktoria; Theorell, Jakob; Friberg, Danielle; Sandberg, Rickard; Mjösberg, Jenny

    2016-04-01

    Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.

  6. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells.

    Science.gov (United States)

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-03-29

    The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.

  7. Novel roles for GlcNAc in cell signaling.

    Science.gov (United States)

    Naseem, Shamoon; Parrino, Salvatore M; Buenten, Dane M; Konopka, James B

    2012-03-01

    N-acetylglucosamine (GlcNAc) has long been known to play important roles in cell surface structure. Recent studies are now revealing new functions for GlcNAc in cell signaling. Exposure to GlcNAc regulates virulence functions in the human fungal pathogen Candida albicans and in pathogenic bacteria. These signaling pathways sense exogenous GlcNAc and are distinct from the O-GlcNAc signaling pathways in mammalian cells in which increased levels of intracellular GlcNAc synthesis leads to post-translational modification of proteins by attachment of O-GlcNAc. The novel roles of GlcNAc in cell signaling will be the subject of this mini-review.

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

    Directory of Open Access Journals (Sweden)

    Dmitri Papatsenko

    2015-08-01

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

  9. A microfluidic platform reveals differential response of regulatory T cells to micropatterned costimulation arrays.

    Science.gov (United States)

    Lee, Joung-Hyun; Dustin, Michael L; Kam, Lance C

    2015-11-01

    -substrate interface, revealing an important role for coincidence of TCR and costimulatory pathway in triggering regulatory T cell function.

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

    Directory of Open Access Journals (Sweden)

    Cecil M Benitez

    2014-10-01

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

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

    Science.gov (United States)

    Benitez, Cecil M; Qu, Kun; Sugiyama, Takuya; Pauerstein, Philip T; Liu, Yinghua; Tsai, Jennifer; Gu, Xueying; Ghodasara, Amar; Arda, H Efsun; Zhang, Jiajing; Dekker, Joseph D; Tucker, Haley O; Chang, Howard Y; Kim, Seung K

    2014-10-01

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

  12. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures.

    Science.gov (United States)

    Ng, Jia-Hui; Kumar, Vibhor; Muratani, Masafumi; Kraus, Petra; Yeo, Jia-Chi; Yaw, Lai-Ping; Xue, Kun; Lufkin, Thomas; Prabhakar, Shyam; Ng, Huck-Hui

    2013-02-11

    The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells.

  13. Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

    DEFF Research Database (Denmark)

    Hattori, Naoko; Niwa, Tohru; Kimura, Kana;

    2013-01-01

    Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which....... Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell...... population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination...

  14. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  15. Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency

    Institute of Scientific and Technical Information of China (English)

    R David Hawkins; Zhen Ye; Samantha Kuan; Pengzhi Yu; Hui Liu; Xinmin Zhang; Roland D Green; Victor V Lobanenkov; Ron Stewart; James A Thomson; Bing Ren; Gary C Hon; Chuhu Yang; Jessica E Antosiewicz-Bourget; LeonardKLee; Que-Minh Ngo; Sarit Klugman; Keith A Ching; Lee E Edsall

    2011-01-01

    Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells.While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types.In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genomewide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage.We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes.We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4mel and H3K27ac,which correlate with expression of their potential target genes.Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs.Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

  16. TLR-induced activation of Btk- Role for endosomal MHC class Ⅱ molecules revealed

    Institute of Scientific and Technical Information of China (English)

    Joan Ni Gabhann; Caroline A Jefferies

    2011-01-01

    @@ MHC molecules have been shown to play key roles in the immune system including regulating T-cell repertoire development through the process of positive and negative selection.MHC molecules also function to bridge the innate and adaptive immune system through the presentation of processed antigenic peptides to T-cells.

  17. Modelling of Yeast Mating Reveals Robustness Strategies for Cell-Cell Interactions.

    Directory of Open Access Journals (Sweden)

    Weitao Chen

    2016-07-01

    Full Text Available Mating of budding yeast cells is a model system for studying cell-cell interactions. Haploid yeast cells secrete mating pheromones that are sensed by the partner which responds by growing a mating projection toward the source. The two projections meet and fuse to form the diploid. Successful mating relies on precise coordination of dynamic extracellular signals, signaling pathways, and cell shape changes in a noisy background. It remains elusive how cells mate accurately and efficiently in a natural multi-cell environment. Here we present the first stochastic model of multiple mating cells whose morphologies are driven by pheromone gradients and intracellular signals. Our novel computational framework encompassed a moving boundary method for modeling both a-cells and α-cells and their cell shape changes, the extracellular diffusion of mating pheromones dynamically coupled with cell polarization, and both external and internal noise. Quantification of mating efficiency was developed and tested for different model parameters. Computer simulations revealed important robustness strategies for mating in the presence of noise. These strategies included the polarized secretion of pheromone, the presence of the α-factor protease Bar1, and the regulation of sensing sensitivity; all were consistent with data in the literature. In addition, we investigated mating discrimination, the ability of an a-cell to distinguish between α-cells either making or not making α-factor, and mating competition, in which multiple a-cells compete to mate with one α-cell. Our simulations were consistent with previous experimental results. Moreover, we performed a combination of simulations and experiments to estimate the diffusion rate of the pheromone a-factor. In summary, we constructed a framework for simulating yeast mating with multiple cells in a noisy environment, and used this framework to reproduce mating behaviors and to identify strategies for robust cell-cell

  18. Integrated metabolomics and transcriptomics reveal enhanced specialized metabolism in Medicago truncatula root border cells.

    Science.gov (United States)

    Watson, Bonnie S; Bedair, Mohamed F; Urbanczyk-Wochniak, Ewa; Huhman, David V; Yang, Dong Sik; Allen, Stacy N; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W

    2015-04-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4'-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4'-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously.

  19. Glucocorticoids play a key role in circadian cell cycle rhythms.

    Directory of Open Access Journals (Sweden)

    Thomas Dickmeis

    2007-04-01

    Full Text Available Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.

  20. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    Science.gov (United States)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  1. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    Science.gov (United States)

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-01

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli.

  2. Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin.

    Science.gov (United States)

    Oh, Jaeho; Edwards, Erin E; McClatchey, P Mason; Thomas, Susan N

    2015-10-15

    Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell-cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner.

  3. Quantification of cell edge velocities and traction forces reveals distinct motility modules during cell spreading.

    Directory of Open Access Journals (Sweden)

    Benjamin J Dubin-Thaler

    Full Text Available Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments -- spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1 During early spreading, cells form initial contacts with the surface. 2 The middle spreading phase exhibits rapidly increasing attachment area. 3 Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters -- a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules

  4. Role of liver stem cells in hepatocarcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Lei-Bo; Xu; Chao; Liu

    2014-01-01

    Liver cancer is an aggressive disease with a high mortality rate. Management of liver cancer is strongly dependent on the tumor stage and underlying liver disease. Unfortunately, most cases are discovered when the cancer is already advanced, missing the opportunity for surgical resection. Thus, an improved understanding of the mechanisms responsible for liver cancer initiation and progression will facilitate the detection of more reliable tumor markers and the development of new small molecules for targeted therapy of liver cancer. Recently, there is increasing evidence for the "cancer stem cell hypothesis", which postulates that liver cancer originates from the malignant transformation of liver stem/progenitor cells(liver cancer stem cells). This cancer stem cell model has important significance for understanding the basic biology of liver cancer and has profound importance for the development of new strategies for cancer prevention and treatment. In this review, we highlight recent advances in the role of liver stem cells in hepatocarcinogenesis. Our review of the literature shows that identification of the cellular origin and the signaling pathways involved is challenging issues in liver cancer with pivotal implications in therapeutic perspectives. Although the dedifferentiation of mature hepatocytes/cholangiocytes in hepatocarcinogenesis cannot be excluded, neoplastic transformation of a stem cell subpopulation more easily explains hepatocarcinogenesis. Elimination of liver cancer stem cells in liver cancer could result in the degeneration of downstream cells, which makes them potential targets for liver cancer therapies. Therefore, liver stem cells could represent a new target for therapeutic approaches to liver cancer in the near future.

  5. Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role.

    Science.gov (United States)

    Hao, Liang; Zhu, Guochun; Lu, Yun; Wang, Min; Jules, Joel; Zhou, Xuedong; Chen, Wei

    2015-05-22

    Using rheumatoid arthritis (RA) and periodontitis mouse models, we demonstrate that RA and periodontitis share many pathological features, such as deregulated cytokine production, increased immune-cell infiltration, increased expression of Toll-like receptors (TLRs), and enhanced osteoclast activity and bone erosion. We reveal that genetic deletion of cathepsin K (Ctsk) caused a radical reduction in inflammation and bone erosion within RA joint capsules and periodontal lesions, a drastic decrease in immune-cell infiltration, and a significant reduction in osteoclasts, macrophages, dendritic and T-cells. Deficiency of Ctsk greatly decreased the expression of TLR-4, 5, and 9 and their downstream cytokines in periodontal gingival epithelial lesions and synovial RA lesions. Hence, Ctsk may be targeted to treat RA and periodontitis simultaneously due to its shared osteoimmune role.

  6. Spinal cord injury reveals multilineage differentiation of ependymal cells.

    Science.gov (United States)

    Meletis, Konstantinos; Barnabé-Heider, Fanie; Carlén, Marie; Evergren, Emma; Tomilin, Nikolay; Shupliakov, Oleg; Frisén, Jonas

    2008-07-22

    Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

  7. Spinal cord injury reveals multilineage differentiation of ependymal cells.

    Directory of Open Access Journals (Sweden)

    Konstantinos Meletis

    2008-07-01

    Full Text Available Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

  8. Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen.

    Science.gov (United States)

    Hayashi, Chie; Papadopoulos, George; Gudino, Cynthia V; Weinberg, Ellen O; Barth, Kenneth R; Madrigal, Andrés G; Chen, Yang; Ning, Hua; LaValley, Michael; Gibson, Frank C; Hamilton, James A; Genco, Caroline A

    2012-10-01

    Clinical and epidemiological studies have implicated chronic infections in the development of atherosclerosis. It has been proposed that common mechanisms of signaling via TLRs link stimulation by multiple pathogens to atherosclerosis. However, how pathogen-specific stimulation of TLR4 contributes to atherosclerosis progression remains poorly understood. In this study, atherosclerosis-prone apolipoprotein-E null (ApoE(-/-)) and TLR4-deficient (ApoE(-/-)TLR4(-/-)) mice were orally infected with the periodontal pathogen Porphyromonas gingivalis. ApoE(-/-)TLR4(-/-) mice were markedly more susceptible to atherosclerosis after oral infection with P. gingivalis. Using live animal imaging, we demonstrate that enhanced lesion progression occurs progressively and was increasingly evident with advancing age. Immunohistochemical analysis of lesions from ApoE(-/-)TLR4(-/-) mice revealed an increased inflammatory cell infiltrate composed primarily of macrophages and IL-17 effector T cells (Th17), a subset linked with chronic inflammation. Furthermore, enhanced atherosclerosis in TLR4-deficient mice was associated with impaired development of Th1 immunity and regulatory T cell infiltration. In vitro studies suggest that the mechanism of TLR4-mediated protective immunity may be orchestrated by dendritic cell IL-12 and IL-10, which are prototypic Th1 and regulatory T cell polarizing cytokines. We demonstrate an atheroprotective role for TLR4 in response to infection with the oral pathogen P. gingivalis. Our results point to a role for pathogen-specific TLR signaling in chronic inflammation and atherosclerosis.

  9. Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells.

    Science.gov (United States)

    Gayraud-Morel, Barbara; Chrétien, Fabrice; Jory, Aurélie; Sambasivan, Ramkumar; Negroni, Elisa; Flamant, Patricia; Soubigou, Guillaume; Coppée, Jean-Yves; Di Santo, James; Cumano, Ana; Mouly, Vincent; Tajbakhsh, Shahragim

    2012-04-01

    Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.

  10. Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

    Directory of Open Access Journals (Sweden)

    Carolina Tropini

    2014-11-01

    Full Text Available Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  11. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

    Science.gov (United States)

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-01-01

    The ‘neural plate border’ of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure. DOI: http://dx.doi.org/10.7554/eLife.21620.001 PMID:28355135

  12. The role of Protein Kinase Cη in T cell biology

    Directory of Open Access Journals (Sweden)

    Nicholas R.J. Gascoigne

    2012-06-01

    Full Text Available Protein kinase Cη (PKCη is a member of the novel PKC subfamily, which also includes δ, ε, and θ isoforms. Compared to the other novel PKCs, the function of PKCη in the immune system is largely unknown. Several studies have started to reveal the role of PKCη, particularly in T cells. PKCη is highly expressed in T cells, and is upregulated during thymocyte positive selection. Interestingly, like the θ isoform, PKCη is also recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell. However, unlike PKCθ, which becomes concentrated to the central region of the synapse, PKCη remains in a diffuse pattern over the whole area of the synapse, suggesting distinctive roles of these two isoforms in signal transduction. Although PKCη is dispensable for thymocyte development, further analysis of PKCη− or PKCθ−deficient and double knockout mice revealed the redundancy of these two isoforms in thymocyte development. In contrast, PKCη rather than PKCθ, plays an important role for T cell homeostatic proliferation, which requires recognition of self-antigen. Another piece of evidence demonstrating that PKCη and PKCθ have isoform specific as well as redundant roles come from the analysis of CD4 to CD8 T cell ratios in the periphery of these knockout mice. Deficiency in PKCη or PKCθ had opposing effects as PKCη knockout mice had a higher ratio of CD4 to CD8 T cells compared to that of wild-type mice, whereas PKCθ-deficient mice had a lower ratio. Biochemical studies showed that calcium flux and NFκB translocation is impaired in PKCη-deficient T cells upon TCR crosslinking stimulation, a character shared with PKCθ-deficient T cells. However, unlike the case with PKCθ, the mechanistic study of PKCη is at early stage and the signaling pathways involving PKCη, at least in T cells, are essentially unknown. In this review, we will cover the topics mentioned above as well as provide some

  13. Adhesion protein networks reveal functions proximal and distal to cell-matrix contacts.

    Science.gov (United States)

    Byron, Adam; Frame, Margaret C

    2016-04-01

    Cell adhesion to the extracellular matrix is generally mediated by integrin receptors, which bind to intracellular adhesion proteins that form multi-molecular scaffolding and signalling complexes. The networks of proteins, and their interactions, are dynamic, mechanosensitive and extremely complex. Recent efforts to characterise adhesions using a variety of technologies, including imaging, proteomics and bioinformatics, have provided new insights into their composition, organisation and how they are regulated, and have also begun to reveal unexpected roles for so-called adhesion proteins in other cellular compartments (for example, the nucleus or centrosomes) in diseases such as cancer. We believe this is opening a new chapter on understanding the wider functions of adhesion proteins, both proximal and distal to cell-matrix contacts.

  14. Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation.

    Directory of Open Access Journals (Sweden)

    Hongkai Ji

    Full Text Available The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP, global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs. We further document that a Myc core signature (MCS set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

  15. Ultrastructural observations reveal the presence of channels between cork cells.

    Science.gov (United States)

    Teixeira, Rita Teresa; Pereira, Helena

    2009-12-01

    The ultrastructure of phellem cells of Quercus suber L. (cork oak) and Calotropis procera (Ait) R. Br. were analyzed using electron transmission microscopy to determine the presence or absence of plasmodesmata (PD). Different types of Q. suber cork samples were studied: one year shoots; virgin cork (first periderm), reproduction cork (traumatic periderm), and wet cork. The channel structures of PD were found in all the samples crossing adjacent cell walls through the suberin layer of the secondary wall. Calotropis phellem also showed PD crossing the cell walls of adjacent cells but in fewer numbers compared to Q. suber. In one year stems of cork oak, it was possible to follow the physiologically active PD with ribosomic accumulation next to the aperture of the channel seen in the phellogen cells to the completely obstructed channels in the dead cells that characterize the phellem tissue.

  16. Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

    Science.gov (United States)

    Le, Anne; Stine, Zachary E.; Nguyen, Christopher; Afzal, Junaid; Sun, Peng; Hamaker, Max; Siegel, Nicholas M.; Gouw, Arvin M.; Kang, Byung-hak; Yu, Shu-Han; Cochran, Rory L.; Sailor, Kurt A.; Song, Hongjun; Dang, Chi V.

    2014-01-01

    Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity. PMID:25114222

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-12

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

  18. Spinal cord injury reveals multilineage differentiation of ependymal cells.

    OpenAIRE

    Konstantinos Meletis; Fanie Barnabé-Heider; Marie Carlén; Emma Evergren; Nikolay Tomilin; Oleg Shupliakov; Jonas Frisén

    2008-01-01

    Author Summary Spinal cord injuries occur in more than 30.000 individuals each year worldwide and result in significant morbidity, with patients requiring long physical and medical care. The recent identification of resident stem cells in the adult spinal cord has opened up for the possibility of pharmacological manipulation of these cells to produce cell types promoting recovery after injury. We have employed genetic tools to specifically address the identity and reaction to injury of a spin...

  19. Poroelasticity of cell nuclei revealed through atomic force microscopy characterization

    Science.gov (United States)

    Wei, Fanan; Lan, Fei; Liu, Bin; Liu, Lianqing; Li, Guangyong

    2016-11-01

    With great potential in precision medical application, cell biomechanics is rising as a hot topic in biology. Cell nucleus, as the largest component within cell, not only contributes greatly to the cell's mechanical behavior, but also serves as the most vital component within cell. However, cell nucleus' mechanics is still far from unambiguous up to now. In this paper, we attempted to characterize and evaluate the mechanical property of isolated cell nuclei using Atomic Force Microscopy with a tipless probe. As indicated from typical indentation, changing loading rate and stress relaxation experiment results, cell nuclei showed significant dynamically mechanical property, i.e., time-dependent mechanics. Furthermore, through theoretical analysis, finite element simulation and stress relaxation experiment, the nature of nucleus' mechanics was better described by poroelasticity, rather than viscoelasticity. Therefore, the essence of nucleus' mechanics was clarified to be poroelastic through a sophisticated analysis. Finally, we estimated the poroelastic parameters for nuclei of two types of cells through a combination of experimental data and finite element simulation.

  20. Identification of ClpP substrates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development.

    Science.gov (United States)

    Bhat, Nowsheen H; Vass, Robert H; Stoddard, Patrick R; Shin, Dong K; Chien, Peter

    2013-06-01

    Energy-dependent proteases ensure the timely removal of unwanted proteins in a highly selective fashion. In Caulobacter crescentus, protein degradation by the ClpXP protease is critical for cell cycle progression; however, only a handful of substrates are currently known. Here, we use a trapping approach to identify putative substrates of the ClpP associated proteases in C. crescentus. Biochemical validation of several of these targets reveals specific protease recognition motifs and suggests a need for ClpXP-specific degradation beyond degradation of known cell cycle regulators. We focus on a particular instance of regulated proteolysis in Caulobacter by exploring the role of ClpXP in degrading the stalk synthesis transcription factor TacA. We show that TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and that stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity. Together, our work reveals a number of new validated ClpXP substrates, clarifies rules of protease substrate selection, and demonstrates how regulated protein degradation is critical for Caulobacter development and cell cycle progression.

  1. Genetic deletion of JAM-C reveals a role in myeloid progenitor generation.

    Science.gov (United States)

    Praetor, Asja; McBride, Jacqueline M; Chiu, Henry; Rangell, Linda; Cabote, Lorena; Lee, Wyne P; Cupp, James; Danilenko, Dimitry M; Fong, Sherman

    2009-02-26

    Hematopoietic stem cells (HSCs) have the capacity to self-renew and continuously differentiate into all blood cell lineages throughout life. At each branching point during differentiation, interactions with the environment are key in the generation of daughter cells with distinct fates. Here, we examined the role of the cell adhesion molecule JAM-C, a protein known to mediate cellular polarity during spermatogenesis, in hematopoiesis. We show that murine JAM-C is highly expressed on HSCs in the bone marrow (BM). Expression correlates with self-renewal, the highest being on long-term repopulating HSCs, and decreases with differentiation, which is maintained longest among myeloid committed progenitors. Inclusion of JAM-C as a sole marker on lineage-negative BM cells yields HSC enrichments and long-term multilineage reconstitution when transferred to lethally irradiated mice. Analysis of Jam-C-deficient mice showed that two-thirds die within 48 hours after birth. In the surviving animals, loss of Jam-C leads to an increase in myeloid progenitors and granulocytes in the BM. Stem cells and myeloid cells from fetal liver are normal in number and homing to the BM. These results provide evidence that JAM-C defines HSCs in the BM and that JAM-C plays a role in controlling myeloid progenitor generation in the BM.

  2. Biophysical characteristics reveal neural stem cell differentiation potential.

    Directory of Open Access Journals (Sweden)

    Fatima H Labeed

    Full Text Available BACKGROUND: Distinguishing human neural stem/progenitor cell (huNSPC populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: We used dielectrophoresis (DEP to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. CONCLUSIONS/SIGNIFICANCE: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

  3. Charge transport in CdTe solar cells revealed by conductive tomographic atomic force microscopy

    Science.gov (United States)

    Luria, Justin; Kutes, Yasemin; Moore, Andrew; Zhang, Lihua; Stach, Eric A.; Huey, Bryan D.

    2016-11-01

    The influence of microstructural defects on the device properties in CdTe remains largely unknown. This is partly because characterization techniques have been unable to image electrical pathways throughout three-dimensional grains and grain boundaries with nanoscale resolution. Here, we employ a conductive and tomographic variation of atomic force microscopy to study charge transport at the nanoscale in a functioning thin-film solar cell with 12.3% efficiency. Images of electric current collected through the device thickness reveal spatially dependent short-circuit and open-circuit performance, and confirm that grain boundaries are preferential pathways for electron transport. Results on samples with and without cadmium chloride treatment reveal little difference in grain structure at the microscale, with samples without treatment showing almost no photocurrent either at planar defects or at grain boundaries. Our results supports an energetically orthogonal transport system of grain boundaries and interconnected planar defects as contributing to optimal solar cell performance, contrary to the conventional wisdom of the deleterious role of planar defects on polycrystalline thin-film solar cells.

  4. Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

    Science.gov (United States)

    Thrower, D A; Bloom, K

    2001-09-01

    We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

  5. Analysis of uncoupling protein 2-deficient mice upon anaesthesia and sedation revealed a role for UCP2 in locomotion.

    Directory of Open Access Journals (Sweden)

    Marie-Clotilde Alves-Guerra

    Full Text Available General anaesthesia is associated with hypothermia, oxidative stress, and immune depression. Uncoupling Protein (UCP2 is a member of the mitochondrial carrier family present in many organs including the spleen, the lung and the brain. A role of UCP2 in the activation of the inflammatory/immune cells, in the secretion of hormones, and in the excitability of neurons by regulating the production of reactive oxygen species has been discussed. Because of the side effects of anaesthesia listed above, we aimed to question the expression and the function of UCP2 during anaesthesia. Induction of anaesthesia with ketamine (20 mg/kg or isoflurane (3.6% and induction of sedation with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg stimulated infiltration of immune cells in the lung and increased UCP2 protein content in the lung, in both immune and non-immune cells. UCP2 content in the lung inversely correlated with body temperature decrease induced by medetomidine treatment. Challenge of the Ucp2(-/- mice with isoflurane and medetomidine revealed an earlier behavioral recovery phenotype. Transponder analysis of body temperature and activity showed no difference between Ucp2(-/- and control mice in basal conditions. However, upon an acute decrease of body temperature induced by medetomidine, Ucp2(-/- mice exhibited increased locomotion activity. Together, these results show that UCP2 is rapidly mobilized during anaesthesia and sedation in immune cells, and suggest a role of UCP2 in locomotion.

  6. Gene pair signatures in cell type transcriptomes reveal lineage control

    Science.gov (United States)

    Heinäniemi, Merja; Nykter, Matti; Kramer, Roger; Wienecke-Baldacchino, Anke; Sinkkonen, Lasse; Zhou, Joseph Xu; Kreisberg, Richard; Kauffman, Stuart A.; Huang, Sui; Shmulevich, Ilya

    2013-01-01

    The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We compiled a resource of curated human expression data comprising 166 cell types and 2,602 transcription regulating genes and developed a data driven method built around the concept of expression reversal defined at the level of gene pairs, such as those participating in toggle switch circuits. This approach allows us to organize the cell types into their ontogenetic lineage-relationships and to reflect regulatory relationships among genes that explain their ability to function as determinants of cell fate. We show that this method identifies genes belonging to regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, thus offering a novel large-scale perspective on lineage specification. PMID:23603899

  7. Plasmacytoid dendritic cell role in cutaneous malignancies.

    Science.gov (United States)

    Saadeh, Dana; Kurban, Mazen; Abbas, Ossama

    2016-07-01

    Plasmacytoid dendritic cells (pDCs) correspond to a specialized dendritic cell population that exhibit plasma cell morphology, express CD4, CD123, HLA-DR, blood-derived dendritic cell antigen-2 (BDCA-2), and Toll-like receptor (TLR)7 and TLR9 within endosomal compartments. Through their production of type I interferons (IFNs) and other pro-inflammatory cytokines, pDCs provide anti-viral resistance and link the innate and adaptive immunity by controlling the function of myeloid DCs, lymphocytes, and natural killer (NK) cells. While lacking from normal skin, pDCs are usually recruited to the skin in several cutaneous pathologies where they appear to be involved in the pathogenesis of several infectious, inflammatory/autoimmune, and neoplastic entities. Among the latter group, pDCs have the potential to induce anti-tumour immunity; however, the complex interaction of pDCs with tumor cells and their micro-environment appears to contribute to immunologic tolerance. In this review, we aim at highlighting the role played by pDCs in cutaneous malignancies with special emphasis on the underlying mechanisms.

  8. Revealed: The spy who regulates neuroblastoma stem cells.

    Science.gov (United States)

    Vora, Parvez; Venugopal, Chitra; Singh, Sheila K

    2014-11-30

    Neuroblastoma (NB), an embryonal tumour of the sympathetic nervous system, is thought to originate from undifferentiated neural crest cells and is known to exhibit extremely heterogeneous biological and clinical behaviors. Occurring in very young children, the median age at diagnosis is 17 months and it accounts for 10% of all pediatric cancer mortalities. The standard treatment regimen for patients with high-risk NB includes induction and surgery followed by isotretinoin or Accutane (13-cis retinoic acid) treatment, which is shown to induce terminal differentiation of NB cells. However, molecular regulators that maintain an undifferentiated phenotype in NB cells are still poorly understood.

  9. Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.

    Directory of Open Access Journals (Sweden)

    Christian Frezza

    Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-15

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

  12. Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres

    Directory of Open Access Journals (Sweden)

    Laura Garcia-Exposito

    2016-11-01

    Full Text Available Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT pathways for telomere maintenance and survival. ALT involves homologous recombination (HR-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID, we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη. We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

  13. Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription

    Science.gov (United States)

    Tavenet, Arounie; Suleau, Audrey; Dubreuil, Géraldine; Ferrari, Roberto; Ducrot, Cécile; Michaut, Magali; Aude, Jean-Christophe; Dieci, Giorgio; Lefebvre, Olivier; Conesa, Christine; Acker, Joël

    2009-01-01

    Human PC4 and the yeast ortholog Sub1 have multiple functions in RNA polymerase II transcription. Genome-wide mapping revealed that Sub1 is present on Pol III-transcribed genes. Sub1 was found to interact with components of the Pol III transcription system and to stimulate the initiation and reinitiation steps in a system reconstituted with all recombinant factors. Sub1 was required for optimal Pol III gene transcription in exponentially growing cells. PMID:19706510

  14. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

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    Yihan Lin

    Full Text Available Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS.

  15. Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo.

    Science.gov (United States)

    Onjiko, Rosemary M; Moody, Sally A; Nemes, Peter

    2015-05-26

    Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.

  16. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.

    Science.gov (United States)

    Invernizzi, F; D'Amato, I; Jensen, P B; Ravaglia, S; Zeviani, M; Tiranti, V

    2012-03-01

    Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

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

    Science.gov (United States)

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

    2016-02-01

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

  18. Mcph1-deficient mice reveal a role for MCPH1 in otitis media.

    Science.gov (United States)

    Chen, Jing; Ingham, Neil; Clare, Simon; Raisen, Claire; Vancollie, Valerie E; Ismail, Ozama; McIntyre, Rebecca E; Tsang, Stephen H; Mahajan, Vinit B; Dougan, Gordon; Adams, David J; White, Jacqueline K; Steel, Karen P

    2013-01-01

    Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1(tm1a) (/tm1a) ) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1(tm1a) (/tm1a) mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1(tm1a) (/tm1a) mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1(tm1a) (/tm1a) mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.

  19. Dissecting a role for melanopsin in behavioural light aversion reveals a response independent of conventional photoreception.

    Directory of Open Access Journals (Sweden)

    Ma'ayan Semo

    Full Text Available Melanopsin photoreception plays a vital role in irradiance detection for non-image forming responses to light. However, little is known about the involvement of melanopsin in emotional processing of luminance. When confronted with a gradient in light, organisms exhibit spatial movements relative to this stimulus. In rodents, behavioural light aversion (BLA is a well-documented but poorly understood phenomenon during which animals attribute salience to light and remove themselves from it. Here, using genetically modified mice and an open field behavioural paradigm, we investigate the role of melanopsin in BLA. While wildtype (WT, melanopsin knockout (Opn4(-/- and rd/rd cl (melanopsin only (MO mice all exhibit BLA, our novel methodology reveals that isolated melanopsin photoreception produces a slow, potentiating response to light. In order to control for the involvement of pupillary constriction in BLA we eliminated this variable with topical atropine application. This manipulation enhanced BLA in WT and MO mice, but most remarkably, revealed light aversion in triple knockout (TKO mice, lacking three elements deemed essential for conventional photoreception (Opn4(-/- Gnat1(-/- Cnga3(-/-. Using a number of complementary strategies, we determined this response to be generated at the level of the retina. Our findings have significant implications for the understanding of how melanopsin signalling may modulate aversive responses to light in mice and humans. In addition, we also reveal a clear potential for light perception in TKO mice.

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

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

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

  1. Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells

    OpenAIRE

    2012-01-01

    Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitat...

  2. Photoactivation approaches reveal a role for Rab11 in FGFR4 recycling and signalling.

    Science.gov (United States)

    Haugsten, Ellen M; Brech, Andreas; Liestøl, Knut; Norman, Jim C; Wesche, Jørgen

    2014-06-01

    Fibroblast growth factor receptor 4 (FGFR4) plays important roles during development and in the adult to maintain tissue homeostasis. Moreover, overexpression of FGFR4 or activating mutations in FGFR4 has been identified as tumour-promoting events in several forms of cancer. Endocytosis is important for regulation of signalling receptors and we have previously shown that FGFR4 is mainly localized to transferrin-positive structures after ligand-induced endocytosis. Here, using a cell line with a defined pericentriolar endocytic recycling compartment, we show that FGFR4 accumulates in this compartment after endocytosis. Furthermore, using classical recycling assays and a new, photoactivatable FGFR4-PA-GFP fusion protein combined with live-cell imaging, we demonstrate that recycling of FGFR4 is dependent on Rab11. Upon Rab11b depletion, FGFR4 is trapped in the pericentriolar recycling compartment and the total levels of FGFR4 in cells are increased. Moreover, fibroblast growth factor 1 (FGF1)-induced autophosphorylation of FGFR4 as well as phosphorylation of phospholipase C (PLC)-γ is prolonged in cells depleted of Rab11. Interestingly, the activation of mitogen-activated protein kinase and AKT pathways were not prolonged but rather reduced in Rab11-depleted cells, indicating that recycling of FGFR4 is important for the nature of its signalling output. Thus, Rab11-dependent recycling of FGFR4 maintains proper levels of FGFR4 in cells and regulates FGF1-induced FGFR4 signalling.

  3. An emerging role of pectic rhamnogalacturonanII for cell wall integrity.

    Science.gov (United States)

    Reboul, Rebecca; Tenhaken, Raimund

    2012-02-01

    The plant cell wall is a complex network of different polysaccharides and glycoproteins, showing high diversity in nature. The essential components, tethering cell wall are under debate, as novel mutants challenge established models. The mutant ugd2,3 with a reduced supply of the important wall precursor UDP-glucuronic acid reveals the critical role of the pectic compound rhamnogalacturonanII for cell wall stability. This polymer seems to be more important for cell wall integrity than the previously favored xyloglucan.

  4. Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo.

    Science.gov (United States)

    Kokkinopoulos, Ioannis; Ishida, Hidekazu; Saba, Rie; Ruchaya, Prashant; Cabrera, Claudia; Struebig, Monika; Barnes, Michael; Terry, Anna; Kaneko, Masahiro; Shintani, Yasunori; Coppen, Steven; Shiratori, Hidetaka; Ameen, Torath; Mein, Charles; Hamada, Hiroshi; Suzuki, Ken; Yashiro, Kenta

    2015-01-01

    In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

  5. Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo.

    Directory of Open Access Journals (Sweden)

    Ioannis Kokkinopoulos

    Full Text Available In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

  6. Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.

    Science.gov (United States)

    Reyes-Reyes, E Merit; Šalipur, Francesca R; Shams, Mitra; Forsthoefel, Matthew K; Bates, Paula J

    2015-08-01

    AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that

  7. Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Sukseree, Supawadee [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Rossiter, Heidemarie; Mildner, Michael [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Pammer, Johannes [Institute of Clinical Pathology, Medical University of Vienna, Vienna (Austria); Buchberger, Maria; Gruber, Florian [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Watanapokasin, Ramida [Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok (Thailand); Tschachler, Erwin [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria); Eckhart, Leopold, E-mail: leopold.eckhart@meduniwien.ac.at [Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna (Austria)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Here we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.

  8. A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.

    Science.gov (United States)

    Franco, Maribel; Seyfried, Nicholas T; Brand, Andrea H; Peng, Junmin; Mayor, Ugo

    2011-05-01

    Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.

  9. Role of Dendritic Cells in Immune Dysfunction

    Science.gov (United States)

    Savary, Cherylyn A.

    1998-01-01

    The specific aims of the project were: (1) Application of the NASA bioreactor to enhance cytokine-regulated proliferation and maturation of dendritic cells (DC). (2) Compare the frequency and function of DC in normal donors and immunocompromised cancer patients. (3) Analyze the effectiveness of cytokine therapy and DC-assisted immunotherapy (using bioreactor-expanded DC) in a murine model of experimental fungal disease. Our investigations have provided new insight into DC immunobiology and have led to the development of methodology to evaluate DC in blood of normal donors and patients. Information gained from these studies has broadened our understanding of possible mechanisms involved in the immune dysfunction of space travelers and earth-bound cancer patients, and could contribute to the design of novel therapies to restore/preserve immunity in these individuals. Several new avenues of investigation were also revealed. The results of studies completed during Round 2 are summarized.

  10. Microfabricated microbial fuel cell arrays reveal electrochemically active microbes.

    Directory of Open Access Journals (Sweden)

    Huijie Hou

    Full Text Available Microbial fuel cells (MFCs are remarkable "green energy" devices that exploit microbes to generate electricity from organic compounds. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications. Hence, research has focused on strategies to enhance the power output of the MFC devices, including exploring more electrochemically active microbes to expand the few already known electricigen families. However, most of the MFC devices are not compatible with high throughput screening for finding microbes with higher electricity generation capabilities. Here, we describe the development of a microfabricated MFC array, a compact and user-friendly platform for the identification and characterization of electrochemically active microbes. The MFC array consists of 24 integrated anode and cathode chambers, which function as 24 independent miniature MFCs and support direct and parallel comparisons of microbial electrochemical activities. The electricity generation profiles of spatially distinct MFC chambers on the array loaded with Shewanella oneidensis MR-1 differed by less than 8%. A screen of environmental microbes using the array identified an isolate that was related to Shewanella putrefaciens IR-1 and Shewanella sp. MR-7, and displayed 2.3-fold higher power output than the S. oneidensis MR-1 reference strain. Therefore, the utility of the MFC array was demonstrated.

  11. Analysis of Uncoupling Protein 2-Deficient Mice upon Anaesthesia and Sedation Revealed a Role for UCP2 in Locomotion

    Science.gov (United States)

    Alves-Guerra, Marie-Clotilde; Aheng, Caroline; Pecqueur, Claire; Masscheleyn, Sandrine; Tharaux, Pierre Louis; Druilhe, Anne; Ricquier, Daniel; Challet, Etienne; Miroux, Bruno

    2012-01-01

    General anaesthesia is associated with hypothermia, oxidative stress, and immune depression. Uncoupling Protein (UCP2) is a member of the mitochondrial carrier family present in many organs including the spleen, the lung and the brain. A role of UCP2 in the activation of the inflammatory/immune cells, in the secretion of hormones, and in the excitability of neurons by regulating the production of reactive oxygen species has been discussed. Because of the side effects of anaesthesia listed above, we aimed to question the expression and the function of UCP2 during anaesthesia. Induction of anaesthesia with ketamine (20 mg/kg) or isoflurane (3.6%) and induction of sedation with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg) stimulated infiltration of immune cells in the lung and increased UCP2 protein content in the lung, in both immune and non-immune cells. UCP2 content in the lung inversely correlated with body temperature decrease induced by medetomidine treatment. Challenge of the Ucp2−/− mice with isoflurane and medetomidine revealed an earlier behavioral recovery phenotype. Transponder analysis of body temperature and activity showed no difference between Ucp2−/− and control mice in basal conditions. However, upon an acute decrease of body temperature induced by medetomidine, Ucp2−/− mice exhibited increased locomotion activity. Together, these results show that UCP2 is rapidly mobilized during anaesthesia and sedation in immune cells, and suggest a role of UCP2 in locomotion. PMID:22900002

  12. Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription.

    Science.gov (United States)

    Xu, Zhixiong; Wei, Gang; Chepelev, Iouri; Zhao, Keji; Felsenfeld, Gary

    2011-03-01

    Insulin (INS) synthesis and secretion from pancreatic β-cells are tightly regulated; their deregulation causes diabetes. Here we map INS-associated loci in human pancreatic islets by 4C and 3C techniques and show that the INS gene physically interacts with the SYT8 gene, located over 300 kb away. This interaction is elevated by glucose and accompanied by increases in SYT8 expression. Inactivation of the INS promoter by promoter-targeting siRNA reduces SYT8 gene expression. SYT8-INS interaction and SYT8 transcription are attenuated by CTCF depletion. Furthermore, SYT8 knockdown decreases insulin secretion in islets. These results reveal a nonredundant role for SYT8 in insulin secretion and indicate that the INS promoter acts from a distance to stimulate SYT8 transcription. This suggests a function for the INS promoter in coordinating insulin transcription and secretion through long-range regulation of SYT8 expression in human islets.

  13. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles.

    Science.gov (United States)

    Julien, Olivier; Zhuang, Min; Wiita, Arun P; O'Donoghue, Anthony J; Knudsen, Giselle M; Craik, Charles S; Wells, James A

    2016-04-05

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

  14. Coordinating Role of RXRα in Downregulating Hepatic Detoxification during Inflammation Revealed by Fuzzy-Logic Modeling.

    Science.gov (United States)

    Keller, Roland; Klein, Marcus; Thomas, Maria; Dräger, Andreas; Metzger, Ute; Templin, Markus F; Joos, Thomas O; Thasler, Wolfgang E; Zell, Andreas; Zanger, Ulrich M

    2016-01-01

    During various inflammatory processes circulating cytokines including IL-6, IL-1β, and TNFα elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXRα playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRα gene silencing versus IL-6 induced negative gene regulation (rs = 0.79; P<0.0001). These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  16. Resolving the role of actoymyosin contractility in cell microrheology.

    Directory of Open Access Journals (Sweden)

    Christopher M Hale

    Full Text Available Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells-stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM and particle-tracking microrheology measurements of living cells.

  17. Localization of Cladosporium fulvum hydrophobins reveals a role for HCf-6 in adhesion

    DEFF Research Database (Denmark)

    Lacroix, Hélène; Whiteford, James; Spanu, Pietro D

    2008-01-01

    Hydrophobins are amphipathic molecules which form part of fungal cell walls and extracellular matrices and perform a variety of roles in fungal growth and development. The tomato pathogen Cladosporium fulvum has six hydrophobin genes, HCf-1 to -6. We have devised an epitope tagging approach...... in that it appears on submerged hyphae. HCf-5 expression is tightly regulated and appears on aerial hyphae early on during growth. Only HCf-1, -3 and -6 were observed during infection; HCf-3 appears on both conidia and emerging germ tubes. We also show that HCf-6 is secreted and coats surfaces under and around...

  18. Comparative transcriptional profiling analysis of the two daughter cells from tobacco zygote reveals the transcriptome differences in the apical and basal cells

    Directory of Open Access Journals (Sweden)

    Hu Tian-Xiang

    2010-08-01

    Full Text Available Abstract Background In angiosperm, after the first asymmetric zygotic cell division, the apical and basal daughter cells follow distinct development pathways. Global transcriptome analysis of these two cells is essential in understanding their developmental differences. However, because of the difficulty to isolate the in vivo apical and basal cells of two-celled proembryo from ovule and ovary in higher plants, the transcriptome analysis of them hasn't been reported. Results In this study, we developed a procedure for isolating the in vivo apical and basal cells of the two-celled proembryo from tobacco (Nicotiana tabacum, and then performed a comparative transcriptome analysis of the two cells by suppression subtractive hybridization (SSH combined with macroarray screening. After sequencing, we identified 797 differentially expressed ESTs corresponding to 299 unigenes. Library sequence analysis successfully identified tobacco homologies of genes involved in embryogenesis and seed development. By quantitative real-time PCR, we validated the differential expression of 40 genes, with 6 transcripts of them specifically expressed in the apical or basal cell. Expression analysis also revealed some transcripts displayed cell specific activation in one of the daughter cells after zygote division. These differential expressions were further validated by in situ hybridization (ISH. Tissue expression pattern analysis also revealed some potential roles of these candidate genes in development. Conclusions The results show that some differential or specific transcripts in the apical and basal cells of two-celled proembryo were successfully isolated, and the identification of these transcripts reveals that these two daughter cells possess distinct transcriptional profiles after zygote division. Further functional work on these differentially or specifically expressed genes will promote the elucidation of molecular mechanism controlling early embryogenesis.

  19. The double-edge role of B cells in mediating antitumor T-cell immunity: Pharmacological strategies for cancer immunotherapy.

    Science.gov (United States)

    Wang, Jing-Zhang; Zhang, Yu-Hua; Guo, Xin-Hua; Zhang, Hong-Yan; Zhang, Yuan

    2016-07-01

    Emerging evidence reveals the controversial role of B cells in antitumor immunity, but the underlying mechanisms have to be explored. Three latest articles published in the issue 521 of Nature in 2015 reconfirmed the puzzling topic and put forward some explanations of how B cells regulate antitumor T-cell responses both positively and negatively. This paper attempts to demonstrate that different B-cell subpopulations have distinct immunological properties and that they are involved in either antitumor responses or immunosuppression. Recent studies supporting the positive and negative roles of B cells in tumor development were summarized comprehensively. Several specific B-cell subpopulations, such as IgG(+), IgA(+), IL-10(+), and regulatory B cells, were described in detail. The mechanisms underlying the controversial B-cell effects were mainly attributed to different B-cell subpopulations, different B-cell-derived cytokines, direct B cell-T cell interaction, different cancer categories, and different malignant stages, and the immunological interaction between B cells and T cells is mediated by dendritic cells. Promising B-cell-based antitumor strategies were proposed and novel B-cell regulators were summarized to present interesting therapeutic targets. Future investigations are needed to make sure that B-cell-based pharmacological strategies benefit cancer immunotherapy substantially.

  20. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

  1. Fluorogenic probes reveal a role of GLUT4 N-glycosylation in intracellular trafficking.

    Science.gov (United States)

    Hirayama, Shinya; Hori, Yuichiro; Benedek, Zsolt; Suzuki, Tadashi; Kikuchi, Kazuya

    2016-10-01

    Glucose transporter 4 (GLUT4) is an N-glycosylated protein that maintains glucose homeostasis by regulating the protein translocation. To date, it has been unclear whether the N-glycan of GLUT4 contributes to its intracellular trafficking. Here, to clarify the role of the N-glycan, we developed fluorogenic probes that label cytoplasmic and plasma-membrane proteins for multicolor imaging of GLUT4 translocation. One of the probes, which is cell impermeant, selectively detected exocytosed GLUT4. Using this probe, we verified the 'log' of the trafficking, in which N-glycan-deficient GLUT4 was transiently translocated to the cell membrane upon insulin stimulation and was rapidly internalized without retention on the cell membrane. The results strongly suggest that the N-glycan functions in the retention of GLUT4 on the cell membrane. This study showed the utility of the fluorogenic probes and indicated that this imaging tool will be applicable for research on various membrane proteins that show dynamic changes in localization.

  2. Role of Prokineticin Receptor-1 in Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Canan G. Nebigil

    2013-06-01

    Full Text Available G protein-coupled receptors (GPCRs form a large class of seven transmembrane (TM domain receptors. The use of endogenous GPCR ligands to activate the stem cell maintenance or to direct cell differentiation would overcome many of the problems currently encountered in the use of stem cells, such as rapid in vitro differentiation and expansion or rejection in clinical applications. This review focuses on the definition of a new GPCR signaling pathway activated by peptide hormones, called “prokineticins”, in epicardium-derived cells (EPDCs. Signaling via prokineticin-2 and its receptor, PKR1, is required for cardiomyocyte survival during hypoxic stress. The binding of prokineticin-2 to PKR1 induces proliferation, migration and angiogenesis in endothelial cells. The expression of prokineticin and PKR1 increases during cardiac remodeling after myocardial infarction. Gain of function of PKR1 in the adult mouse heart revealed that cardiomyocyte-PKR1 signaling activates EPDCs in a paracrine fashion, thereby promoting de novo vasculogenesis. Transient PKR1 gene therapy after myocardial infarction in mice decreases mortality and improves heart function by promoting neovascularization, protecting cardiomyocytes and mobilizing WT1+ cells. Furthermore, PKR1 signaling promotes adult EPDC proliferation and differentiation to adopt endothelial and smooth muscle cell fate, for the induction of de novo vasculogenesis. PKR1 is expressed in the proepicardium and epicardial cells derived from mice kidneys. Loss of PKR1 causes deficits in EPDCs in the neonatal mice hearts and kidneys and impairs vascularization and heart and kidney function. Taken together, these data indicate a novel role for PKR1 in heart-kidney complex via EPDCs.

  3. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    Science.gov (United States)

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  4. The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis

    Science.gov (United States)

    Boehm, Cordula M.; Obado, Samson; Gadelha, Catarina; Kaupisch, Alexandra; Manna, Paul T.; Gould, Gwyn W.; Rout, Michael P.; Field, Mark C.

    2017-01-01

    Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities. PMID:28114397

  5. A Shotgun Proteomics Approach Reveals a New Toxic Role For Alzheimer's Disease Aβ Peptide: Spliceosome Impairment.

    Science.gov (United States)

    Nuzzo, Domenico; Inguglia, Luigi; Walters, Jessica; Picone, Pasquale; Di Carlo, Marta

    2017-02-03

    Proteomic changes have been described in many neurodegenerative diseases including Alzheimer's disease (AD). However, the early events in the onset of the pathology are yet to be fully elucidated. A cell model system in which LAN5 neuroblastoma cells were incubated for a short time with a recombinant form of Aβ42 (rAβ42) was utilized. Proteins extracted from these cells were subjected to shotgun proteomics analysis by LTQ-Orbitrap-MS followed by label-free quantitation. By bioinformatics tools we found that the most significant of those found to be up-regulated were related to cytoskeletal dynamics (Rho related) and membrane related processes. The most significant of the down-regulated proteins were hnRNP-related. In particular hnRNPs involved in ribosomal biogenesis and in splicing were down-regulated. The latter of these processes stood out as it was highlighted ubiquitously and with the highest significance in the results of every analysis. Furthermore, our findings revealed downregulation at every stage of the splicing process through down-regulation of every subunit of the spliceosome. Dysregulation of the spliceosome was also confirmed using a Western blot ì In conclusion, these data suggest dysregulation of the proteins and processes identified as early events in pathogenesis of AD following Aβ accumulation.

  6. A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

    Directory of Open Access Journals (Sweden)

    Karen M Chisholm

    Full Text Available Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-18

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

  8. In silico synchronization reveals regulators of nuclear ruptures in lamin A/C deficient model cells

    Science.gov (United States)

    Robijns, J.; Molenberghs, F.; Sieprath, T.; Corne, T. D. J.; Verschuuren, M.; de Vos, W. H.

    2016-07-01

    The nuclear lamina is a critical regulator of nuclear structure and function. Nuclei from laminopathy patient cells experience repetitive disruptions of the nuclear envelope, causing transient intermingling of nuclear and cytoplasmic components. The exact causes and consequences of these events are not fully understood, but their stochastic occurrence complicates in-depth analyses. To resolve this, we have established a method that enables quantitative investigation of spontaneous nuclear ruptures, based on co-expression of a firmly bound nuclear reference marker and a fluorescent protein that shuttles between the nucleus and cytoplasm during ruptures. Minimally invasive imaging of both reporters, combined with automated tracking and in silico synchronization of individual rupture events, allowed extracting information on rupture frequency and recovery kinetics. Using this approach, we found that rupture frequency correlates inversely with lamin A/C levels, and can be reduced in genome-edited LMNA knockout cells by blocking actomyosin contractility or inhibiting the acetyl-transferase protein NAT10. Nuclear signal recovery followed a kinetic that is co-determined by the severity of the rupture event, and could be prolonged by knockdown of the ESCRT-III complex component CHMP4B. In conclusion, our approach reveals regulators of nuclear rupture induction and repair, which may have critical roles in disease development.

  9. Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV.

    Directory of Open Access Journals (Sweden)

    Anneleen Hombrouck

    2007-03-01

    Full Text Available Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75-integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.

  10. Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection.

    Science.gov (United States)

    Misas-Villamil, Johana C; Toenges, Gerrit; Kolodziejek, Izabella; Sadaghiani, Amir M; Kaschani, Farnusch; Colby, Thomas; Bogyo, Matthew; van der Hoorn, Renier A L

    2013-02-01

    Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

  11. Targeting cancer stem cells: emerging role of Nanog transcription factor

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    Wang ML

    2013-09-01

    Full Text Available Mong-Lien Wang,1 Shih-Hwa Chiou,2,3 Cheng-Wen Wu1,4–61Institute of Biochemistry and Molecular Biology, 2Institute of Pharmacology, National Yang Ming University, Taipei, Taiwan; 3Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Institute of Microbiology and Immunology, 5Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan; 6Institute of Biomedical Science, Academia Sinica, Taipei, TaiwanAbstract: The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the

  12. Separable roles of UFO during floral development revealed by conditional restoration of gene function.

    Science.gov (United States)

    Laufs, Patrick; Coen, Enrico; Kronenberger, Jocelyne; Traas, Jan; Doonan, John

    2003-02-01

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.

  13. Arabidopsis transcriptome analysis reveals key roles of melatonin in plant defense systems.

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    Sarah Weeda

    Full Text Available Melatonin is a ubiquitous molecule and exists across kingdoms including plant species. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. Much less attention has been drawn to its affect on genome-wide gene expression. To comprehensively investigate the role(s of melatonin at the genomics level, we utilized mRNA-seq technology to analyze Arabidopsis plants subjected to a 16-hour 100 pM (low and 1 mM (high melatonin treatment. The expression profiles were analyzed to identify differentially expressed genes. 100 pM melatonin treatment significantly affected the expression of only 81 genes with 51 down-regulated and 30 up-regulated. However, 1 mM melatonin significantly altered 1308 genes with 566 up-regulated and 742 down-regulated. Not all genes altered by low melatonin were affected by high melatonin, indicating different roles of melatonin in regulation of plant growth and development under low and high concentrations. Furthermore, a large number of genes altered by melatonin were involved in plant stress defense. Transcript levels for many stress receptors, kinases, and stress-associated calcium signals were up-regulated. The majority of transcription factors identified were also involved in plant stress defense. Additionally, most identified genes in ABA, ET, SA and JA pathways were up-regulated, while genes pertaining to auxin responses and signaling, peroxidases, and those associated with cell wall synthesis and modifications were mostly down-regulated. Our results indicate critical roles of melatonin in plant defense against various environmental stresses, and provide a framework for functional analysis of genes in melatonin-mediated signaling pathways.

  14. Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

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    Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; LiyunZhong

    2017-04-01

    Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

  15. Role of viruses in renal cell carcinoma

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    Mehdi Salehipoor

    2012-01-01

    Full Text Available To determine whether viral infections are related to renal cell carcinoma (RCC, we studied 49 patients with RCC (29 patients were males with age ranging from 30 to 81 years and a mean of 57.5 years; 20 patients were females with age ranging from 36 to 70 years with a mean of 58.4 years and 16 non-neoplastic kidney patients as controls. Tissues specimens from study patients and controls were examined by nested polymerase chain reaction (PCR to determine the presence of DNA of several viruses including human papilloma virus (HPV, Epstein-Barr virus (EBV, and polyoma viruses (BKV and JCV. Our results revealed that 7 of 49 (14.29% RCC tissue specimens had HPV DNA compared with none of 16 non-cancer control subjects. Regarding the HPV types, all the positive results were high-risk HPV types (type 16 in three and 18 in four patients. The present study suggests that HPV infection, especially high-risk types, is associated with RCC. However, more studies are necessary to demonstrate the molecular oncogenic processes involved in this association.

  16. Transcriptional dynamics reveal critical roles for non-coding RNAs in the immediate-early response.

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    Stuart Aitken

    2015-04-01

    Full Text Available The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

  17. [Molecular mechanism maintaining muscle satellite cells and the roles in sarcopenia.

    Science.gov (United States)

    Takemoto, Yusei; Fukada, So-Ichiro

    2017-01-01

    Skeletal muscle has its stem cell named satellite cell. The absence of satellite cells does not allow muscle regeneration, it is unquestionable that satellite cell is indispensable for muscle regeneration processes. A certain number of satellite cells appear to be necessary for the successful muscle regeneration, meaning the maintenance of the satellite cells is essential for the functional homeostasis of skeletal muscle. Recent studies have revealed the molecular mechanism underlying satellite cell maintenance in a steady state. A loss of those molecules responsible for the maintenance often results in decreased satellite cell pool and reduced regeneration ability. On the other hand, the contribution of satellite cells to muscle hypertrophy or aged-related atrophy(sarcopenia)is controversial. In this review, we will introduce the molecules that regulate satellite cells homeostasis in the dormant state and then further discuss the recent results on the roles of satellite cell in sarcopenia.

  18. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.

  19. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium.

    Science.gov (United States)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M; Nguyen, Amelia Y; Gritsenko, Marina A; Smith, Richard D; Koppenaal, David W; Pakrasi, Himadri B

    2016-06-01

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram

  20. Role of T Cells in Malnutrition and Obesity

    OpenAIRE

    Gerriets, Valerie A.; MacIver, Nancie J.

    2014-01-01

    Nutritional status is critically important for immune cell function. While obesity is characterized by inflammation that promotes metabolic syndrome including cardiovascular disease and insulin resistance, malnutrition can result in immune cell defects and increased risk of mortality from infectious diseases. T cells play an important role in the immune adaptation to both obesity and malnutrition. T cells in obesity have been shown to have an early and critical role in inducing inflammation, ...

  1. Ascl3 knockout and cell ablation models reveal complexity of salivary gland maintenance and regeneration.

    Science.gov (United States)

    Arany, Szilvia; Catalán, Marcelo A; Roztocil, Elisa; Ovitt, Catherine E

    2011-05-15

    Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3(EGFP-Cre/+) knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.

  2. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

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    Jyuhn-Huarng Juang

    2015-02-01

    Full Text Available The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

  3. Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons.

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    Liviu Aron

    Full Text Available The mechanisms underlying the selective death of substantia nigra (SN neurons in Parkinson disease (PD remain elusive. While inactivation of DJ-1, an oxidative stress suppressor, causes PD, animal models lacking DJ-1 show no overt dopaminergic (DA neuron degeneration in the SN. Here, we show that aging mice lacking DJ-1 and the GDNF-receptor Ret in the DA system display an accelerated loss of SN cell bodies, but not axons, compared to mice that only lack Ret signaling. The survival requirement for DJ-1 is specific for the GIRK2-positive subpopulation in the SN which projects exclusively to the striatum and is more vulnerable in PD. Using Drosophila genetics, we show that constitutively active Ret and associated Ras/ERK, but not PI3K/Akt, signaling components interact genetically with DJ-1. Double loss-of-function experiments indicate that DJ-1 interacts with ERK signaling to control eye and wing development. Our study uncovers a conserved interaction between DJ-1 and Ret-mediated signaling and a novel cell survival role for DJ-1 in the mouse. A better understanding of the molecular connections between trophic signaling, cellular stress and aging could uncover new targets for drug development in PD.

  4. Genetic analysis reveals an unexpected role of BMP7 in initiation of ureteric bud outgrowth in mouse embryos.

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    Alexandre Gonçalves

    Full Text Available BACKGROUND: Genetic analysis in the mouse revealed that GREMLIN1 (GREM1-mediated antagonism of BMP4 is essential for ureteric epithelial branching as the disruption of ureteric bud outgrowth and renal agenesis in Grem1-deficient embryos is restored by additional inactivation of one Bmp4 allele. Another BMP ligand, BMP7, was shown to control the proliferative expansion of nephrogenic progenitors and its requirement for nephrogenesis can be genetically substituted by Bmp4. Therefore, we investigated whether BMP7 in turn also participates in inhibiting ureteric bud outgrowth during the initiation of metanephric kidney development. METHODOLOGY/PRINCIPAL FINDINGS: Genetic inactivation of one Bmp7 allele in Grem1-deficient mouse embryos does not alleviate the bilateral renal agenesis, while complete inactivation of Bmp7 restores ureteric bud outgrowth and branching. In mouse embryos lacking both Grem1 and Bmp7, GDNF/WNT11 feedback signaling and the expression of the Etv4 target gene, which regulates formation of the invading ureteric bud tip, are restored. In contrast to the restoration of ureteric bud outgrowth and branching, nephrogenesis remains aberrant as revealed by the premature loss of Six2 expressing nephrogenic progenitor cells. Therefore, very few nephrons develop in kidneys lacking both Grem1 and Bmp7 and the resulting dysplastic phenotype is indistinguishable from the one of Bmp7-deficient mouse embryos. CONCLUSIONS/SIGNIFICANCE: Our study reveals an unexpected inhibitory role of BMP7 during the onset of ureteric bud outgrowth. As BMP4, BMP7 and GREM1 are expressed in distinct mesenchymal and epithelial domains, the localized antagonistic interactions of GREM1 with BMPs could restrict and guide ureteric bud outgrowth and branching. The robustness and likely significant redundancy of the underlying signaling system is evidenced by the fact that global reduction of Bmp4 or inactivation of Bmp7 are both able to restore ureteric bud outgrowth

  5. Anatomical and biochemical analysis reveal the role of anthocyanins in flower coloration of herbaceous peony.

    Science.gov (United States)

    Zhao, Da-Qiu; Wei, Meng-Ran; Liu, Ding; Tao, Jun

    2016-05-01

    Herbaceous peony (Paeonia lactiflora Pall.) is particularly appreciated because of its elegant and gorgeous flower color, but little is known about the underlying mechanisms of flower coloration. In this study, three P. lactiflora cultivars 'Xuefeng', 'Fenyulou' and 'Dahonglou' with white, pink and red flower were selected as the materials. Their anatomical structures, cell sap pH and metal elements were investigated, and the colored pigment mainly distributed in palisade mesophyll was only found in 'Fenyulou' and 'Dahonglou', and their shape of epidermal cells, cell sap pH and metal elements were not the key factors deciding phenotype color. Moreover, the qualitative and quantitative analysis of flavonoids were performed, their total anthocyanin, anthoxanthin and flavonoid contents were decreased during flower development, and only anthocyanin content in 'Dahonglou' was always higher than that in 'Xuefeng' and 'Fenyulou'. Subsequently, three anthocyanin compositions were found, and peonidin 3,5-di-O-glucoside (Pn3G5G) was identified as the main anthocyanin composition. In addition, the full-length of flavonol synthase gene (FLS) was isolated with the GenBank accession number KM259902, and the expression patterns of eight flavonoid biosynthetic genes showed that only PlDFR and PlANS basically had the highest levels in 'Dahonglou' and the lowest levels in 'Xuefeng', and they basically displayed a descended trend during flower development especially PlDFR, suggesting that these two genes might play a key role in the anthocyanin biosynthesis which resulted in the shift from white to pink and red in flowers. These results would contribute to understand the underlying molecular mechanisms of flower coloration in P. lactiflora.

  6. RNA interference revealed the roles of two carboxylesterase genes in insecticide detoxification in Locusta migratoria.

    Science.gov (United States)

    Zhang, Jianqin; Li, Daqi; Ge, Pingting; Yang, Meiling; Guo, Yaping; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2013-10-01

    Carboxylesterases (CarEs) play key roles in metabolism of specific hormones and detoxification of dietary and environmental xenobiotics in insects. We sequenced and characterized CarE cDNAs putatively derived from two different genes named LmCesA1 and LmCesA2 from the migratory locust, Locusta migratoria, one of the most important agricultural pests in the world. The full-length cDNAs of LmCesA1 (1892 bp) and LmCesA2 (1643 bp) encode 543 and 501 amino acid residues, respectively. The two deduced CarEs share a characteristic α/β-hydrolase structure, including a catalytic triad composed of Ser-Glu (Asp)-His and a consensus sequence GQSAG, which suggests that both CarEs are biologically active. Phylogenetic analysis grouped both LmCesA1 and LmCesA2 into clade A which has been suggested to be involved in dietary detoxification. Both transcripts were highly expressed in all the nymphal and adult stages, but only slightly expressed in eggs. Analyses of tissue-dependent expression and in situ hybridization revealed that both transcripts were primarily expressed in gastric caeca. RNA interference (RNAi) of LmCesA1 and LmCesA2 followed by a topical application of carbaryl or deltamethrin did not lead to a significantly increased mortality with either insecticide. However, RNAi of LmCesA1 and LmCesA2 increased insect mortalities by 20.9% and 14.5%, respectively, when chlorpyrifos was applied. These results suggest that these genes might not play a significant role in detoxification of carbaryl and deltamethrin but are most likely to be involved in detoxification of chlorpyrifos in L. migratoria.

  7. Cross-Talk between CLL Cells and Bone Marrow Endothelial Cells: Role of Signal Transducer and Activator of Transcription-3

    Science.gov (United States)

    Badoux, Xavier; Bueso-Ramos, Carlos; Harris, David; Li, Ping; Liu, Zhiming; Burger, Jan; O’Brien, Susan; Ferrajoli, Alessandra; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Summary Chronic lymphocytic leukemia (CLL) bone marrow is characterized by increased angiogenesis. However, the molecular mediators of neovascularization and the biological significance of increased endothelial cell proliferation in CLL require further investigation. Because signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL we studied the role of STAT3 in modulating vascular endothelial growth factor (VEGF) expression and the effect of vascular endothelial cells on CLL cells. Using chromatin immunoprecipitation (ChIP) we found that anti-STAT3 antibodies immunoprecipitated DNA of STAT3, VEGF and other STAT3-regulated genes. In addition, STAT3-short interfering RNA significantly reduced mRNA levels of VEGF in CLL cells suggesting that STAT3 induces VEGF expression in CLL. Remarkably, bone marrow CLL cells expressed high levels of VEGF and high VEGF levels were detected in the plasma of patients with untreated CLL and correlated with white blood cell count. CLL bone marrow biopsies revealed increased microvascular density and attachment of CLL cells to endothelial cells. Co-culture of CLL and human umbilical vein endothelial cells (HUVEC) cells showed a similar attachment. Furthermore, co-culture studies with HUVEC showed that HUVEC protected CLL cells from spontaneous apoptosis by direct cell-to-cell contact as assessed by flow cytometry using Annexin V. Our data suggest that constitutively activated STAT3 induces VEGF production by CLL cells and CLL cells derive a survival advantage from endothelial cells via cell-to cell contact. PMID:21733558

  8. The Roles of Regulatory B Cells in Cancer

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    Yan He

    2014-01-01

    Full Text Available Regulatory B cells (Bregs, a newly described subset of B cells, have been proved to play a suppressive role in immune system. Bregs can inhibit other immune cells through cytokines secretion and antigen presentation, which give them the role in the pathogenesis of autoimmune diseases and cancers. There are no clear criteria to identify Bregs; different markers were used in the different experimental conditions. Massive researches had described the functions of immune cells such as regulatory T cells (Tregs, dendritic cells (DCs, and B cells in the autoimmune disorder diseases and cancers. More and more researches focused on the roles of Bregs and the cytokines such as Interleukin-10 (IL-10 and transforming growth factor beta (TGF-β secreted by Bregs. The aim of this review is to summarize the characteristics of Bregs and the roles of Bregs in cancer.

  9. The Pointing Errors in Optic Ataxia Reveal the Role of "Peripheral Magnification" of the PPC.

    Science.gov (United States)

    Vindras, Philippe; Blangero, Annabelle; Ota, Hisaaki; Reilly, Karen T; Rossetti, Yves; Pisella, Laure

    2016-01-01

    Interaction with visual objects in the environment requires an accurate correspondence between visual space and its internal representation within the brain. Many clinical conditions involve some impairment in visuo-motor control and the errors created by the lesion of a specific brain region are neither random nor uninformative. Modern approaches to studying the neuropsychology of action require powerful data-driven analyses and error modeling in order to understand the function of the lesioned areas. In the present paper we carried out mixed-effect analyses of the pointing errors of seven optic ataxia patients and seven control subjects. We found that a small parameter set is sufficient to explain the pointing errors produced by unilateral optic ataxia patients. In particular, the extremely stereotypical errors made when pointing toward the contralesional visual field can be fitted by mathematical models similar to those used to model central magnification in cortical or sub-cortical structure(s). Our interpretation is that visual areas that contain this footprint of central magnification guide pointing movements when the posterior parietal cortex (PPC) is damaged and that the functional role of the PPC is to actively compensate for the under-representation of peripheral vision that accompanies central magnification. Optic ataxia misreaching reveals what would be hand movement accuracy and precision if the human motor system did not include elaborated corrective processes for reaching and grasping to non-foveated targets.

  10. Potential Role of the Last Half Repeat in TAL Effectors Revealed by a Molecular Simulation Study

    Directory of Open Access Journals (Sweden)

    Hua Wan

    2016-01-01

    Full Text Available TAL effectors (TALEs contain a modular DNA-binding domain that is composed of tandem repeats. In all naturally occurring TALEs, the end of tandem repeats is invariantly a truncated half repeat. To investigate the potential role of the last half repeat in TALEs, we performed comparative molecular dynamics simulations for the crystal structure of DNA-bound TALE AvrBs3 lacking the last half repeat and its modeled structure having the last half repeat. The structural stability analysis indicates that the modeled system is more stable than the nonmodeled system. Based on the principle component analysis, it is found that the AvrBs3 increases its structural compactness in the presence of the last half repeat. The comparison of DNA groove parameters of the two systems implies that the last half repeat also causes the change of DNA major groove binding efficiency. The following calculation of hydrogen bond reveals that, by stabilizing the phosphate binding with DNA at the C-terminus, the last half repeat helps to adopt a compact conformation at the protein-DNA interface. It further mediates more contacts between TAL repeats and DNA nucleotide bases. Finally, we suggest that the last half repeat is required for the high-efficient recognition of DNA by TALE.

  11. Potential Role of the Last Half Repeat in TAL Effectors Revealed by a Molecular Simulation Study

    Science.gov (United States)

    Wan, Hua; Chang, Shan; Hu, Jian-ping; Tian, Xu-hong

    2016-01-01

    TAL effectors (TALEs) contain a modular DNA-binding domain that is composed of tandem repeats. In all naturally occurring TALEs, the end of tandem repeats is invariantly a truncated half repeat. To investigate the potential role of the last half repeat in TALEs, we performed comparative molecular dynamics simulations for the crystal structure of DNA-bound TALE AvrBs3 lacking the last half repeat and its modeled structure having the last half repeat. The structural stability analysis indicates that the modeled system is more stable than the nonmodeled system. Based on the principle component analysis, it is found that the AvrBs3 increases its structural compactness in the presence of the last half repeat. The comparison of DNA groove parameters of the two systems implies that the last half repeat also causes the change of DNA major groove binding efficiency. The following calculation of hydrogen bond reveals that, by stabilizing the phosphate binding with DNA at the C-terminus, the last half repeat helps to adopt a compact conformation at the protein-DNA interface. It further mediates more contacts between TAL repeats and DNA nucleotide bases. Finally, we suggest that the last half repeat is required for the high-efficient recognition of DNA by TALE. PMID:27803930

  12. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue

    Directory of Open Access Journals (Sweden)

    Kevin M. Harlen

    2016-06-01

    Full Text Available Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II C-terminal domain (CTD and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7, we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3′ end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3′ splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

  13. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

    Directory of Open Access Journals (Sweden)

    Rebecca Yee

    2015-12-01

    Full Text Available Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus.

  14. Role of inositol phospholipid signaling in natural killer cell biology

    Directory of Open Access Journals (Sweden)

    Matthew eGumbleton

    2013-03-01

    Full Text Available Natural Killer (NK cells are important in the host defense against malignancy and infection. At a cellular level NK cells are activated when signals from activating receptors exceed signaling from inhibitory receptors. At a molecular level NK cells undergo an education process to prevent autoimmunity. Mouse models have shown important roles for inositol phospholipid signaling in lymphocytes. NK cells from mice with deletion in different members of the PI3K signaling pathway have defective development, natural killer cell repertoire expression (NKRR and effector function. Here we review the role of inositol phospholipid signaling in NK cell biology.

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

    Science.gov (United States)

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

    2016-07-26

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

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

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-09

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

  18. Cell cycle synchronization reveals greater G2/M-phase accumulation of lung epithelial cells exposed to titanium dioxide nanoparticles.

    Science.gov (United States)

    Medina-Reyes, Estefany I; Bucio-López, Laura; Freyre-Fonseca, Verónica; Sánchez-Pérez, Yesennia; García-Cuéllar, Claudia M; Morales-Bárcenas, Rocío; Pedraza-Chaverri, José; Chirino, Yolanda I

    2015-03-01

    Titanium dioxide has been classified in the 2B group as a possible human carcinogen by the International Agency for Research on Cancer, and amid concerns of its exposure, cell cycle alterations are an important one. However, several studies show inconclusive effects, mainly because it is difficult to compare cell cycle effects caused by TiO2 nanoparticle (NP) exposure between different shapes and sizes of NP, cell culture types, and time of exposure. In addition, cell cycle is frequently analyzed without cell cycle synchronization, which may also mask some effects. We hypothesized that synchronization after TiO2 NP exposure could reveal dissimilar cell cycle progression when compared with unsynchronized cell population. To test our hypothesis, we exposed lung epithelial cells to 1 and 10 μg/cm(2) TiO2 NPs for 7 days and one population was synchronized by serum starvation and inhibition of ribonucleotide reductase using hydroxyurea. Another cell population was exposed to TiO2 NPs under the same experimental conditions, but after treatments, cell cycle was analyzed without synchronization. Our results showed that TiO2 NP-exposed cells without synchronization had no changes in cell cycle distribution; however, cell population synchronized after 1 and 10 μg/cm(2) TiO2 NP treatment showed a 1.5-fold and 1.66-fold increase, respectively, in proliferation. Synchronized cells also reveal a faster capability of TiO2 NP-exposed cells to increase cell population in the G2/M phase in the following 9 h after synchronization. We conclude that synchronization discloses a greater percentage of cells in the G2/M phase and higher proliferation than TiO2 NP-synchronized cells.

  19. Dendritic cells and their role in tumor immunosurveillance

    NARCIS (Netherlands)

    Strioga, M.M.; Schijns, V.E.J.C.; Powell, D.J.; Pasukoniene, V.; Dobrovolskiene, N.T.; Michalek, J.

    2013-01-01

    Dendritic cells (DCs) comprise a heterogeneous population of cells that play a key role in initiating, directing and regulating adaptive immune responses, including those critically involved in tumor immunosurveillance. As a riposte to the central role of DCs in the generation of antitumor immune re

  20. Dental pulp stem cells differentiation reveals new insights in Oct4A dynamics.

    Directory of Open Access Journals (Sweden)

    Federico Ferro

    Full Text Available Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC. A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A. Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.

  1. Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

    Science.gov (United States)

    Lathia, J D; Hitomi, M; Gallagher, J; Gadani, S P; Adkins, J; Vasanji, A; Liu, L; Eyler, C E; Heddleston, J M; Wu, Q; Minhas, S; Soeda, A; Hoeppner, D J; Ravin, R; McKay, R D G; McLendon, R E; Corbeil, D; Chenn, A; Hjelmeland, A B; Park, D M; Rich, J N

    2011-09-01

    Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

  2. Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states

    DEFF Research Database (Denmark)

    Hall, V. J.; Jacobsen, Janus Valentin; Rasmussen, M. A.

    2010-01-01

    Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing...... pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have...

  3. Transcriptome Analysis of Pepper (Capsicum annuum Revealed a Role of 24-epibrassinolide in Response to Chilling

    Directory of Open Access Journals (Sweden)

    Li Jie

    2016-08-01

    Full Text Available Brassinosteroids (BRs have positive effects on many processes during plant growth, development, and various abiotic stress responses. However, little information is available regarding the global gene expression of BRs in response to chilling stress in pepper. In this study, we used RNA sequencing to determine the molecular roles of 24-epibrassinolide (EBR during a chilling stress response. There were 39,829 transcripts, and, among them, 656 were differently-expressed genes (DEGs following EBR treatment (Chill+EBR compared with the control (Chill only, including 335 up-regulated and 321 down-regulated DEGs. We selected 20 genes out of the 656 DEGs for RT-qPCR analysis to confirm the RNA-Seq. Based on GO enrich and KEGG pathway analysis, we found that photosynthesis was significantly up-enriched in biological processes, accompanied by significant increases in the net photosynthetic rate (Pn, Fv/Fm and chlorophyll content. Furthermore, the results indicate that EBR enhanced endogenous levels of salicylic acid (SA and jasmonic acid (JA while suppressing the ethylene (ETH biosynthesis pathway, suggesting that BRs function via a synergistic cross-talk with SA, JA and ETH signaling pathways in response to chilling stress. In addition, EBR induced cellulose synthase-like protein and UDP-glycosyltransferase, suggesting a contribution to the formation of cell wall and hormone metabolism. EBR also triggered the calcium signaling transduction in cytoplasm, and activated the expression of cellular redox homeostasis related genes, such as GSTX1, PER72, and CAT2. This work, therefor, identified the specific genes showed different expression patterns in EBR-treated pepper and associated with the processes of hormone metabolism, redox, signaling, transcription and defense. Our study provides the first evidence of the potent roles of BRs, at the transcription level, to induce the tolerance to chilling stress in pepper as a function of the combination of the

  4. Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.

    Science.gov (United States)

    Baubec, Tuncay; Colombo, Daniele F; Wirbelauer, Christiane; Schmidt, Juliane; Burger, Lukas; Krebs, Arnaud R; Akalin, Altuna; Schübeler, Dirk

    2015-04-09

    DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

  5. Comprehensive expression profiling of rice tetraspanin genes reveals diverse roles during development and abiotic stress

    Directory of Open Access Journals (Sweden)

    Balaji eM

    2015-12-01

    Full Text Available Tetraspanin family is comprised of evolutionarily conserved integral membrane proteins. The incredible ability of tetraspanins to form ‘micro domain complexes’ and their preferential targeting to membranes emphasizes their active association with signal recognition and communication with neighboring cells, thus acting as key modulators of signaling cascades. In animals, tetraspanins are associated with multitude of cellular processes. Unlike animals, the biological relevance of tetraspanins in plants has not been well investigated. In Arabidopsis tetraspanins are known to contribute in important plant development processes such as leaf morphogenesis, root and floral organ formation. In the present study we investigated the genomic organization, chromosomal distribution, phylogeny and domain structure of 15 rice tetraspanin proteins (OsTETs. OsTET proteins had similar domain structure and signature ‘GCCK/R’ motif as reported in Arabidopsis. Comprehensive expression profiling of OsTET genes suggested their possible involvement during rice development. While OsTET9 and 10 accumulated predominantly in flowers, OsTET5, 8 and 12 were preferentially expressed in root tissues. Noticeably, seven OsTETs exhibited more than 2-fold up regulation at early stages of flag leaf senescence in rice. Furthermore, several OsTETs were differentially regulated in rice seedlings exposed to abiotic stresses, exogenous treatment of hormones and nutrient deprivation. Transient subcellular localization studies of eight OsTET proteins in tobacco epidermal cells showed that these proteins localized in plasma membrane. The present study provides valuable insights into the possible roles of tetraspanins in regulating development and defining response to abiotic stresses in rice. Targeted proteomic studies would be useful in identification of their interacting partners under different conditions and ultimately their biological function in plants

  6. Role of T cells in malnutrition and obesity

    Directory of Open Access Journals (Sweden)

    Valerie A Gerriets

    2014-08-01

    Full Text Available Nutritional status is critically important for immune cell function. While obesity is characterized by inflammation that promotes metabolic syndrome including cardiovascular disease and insulin resistance, malnutrition can result in immune cell defects and increased risk of mortality from infectious diseases. T cells play an important role in the immune adaptation to both obesity and malnutrition. T cells in obesity have been shown to have an early and critical role in inducing inflammation, accompanying the accumulation of inflammatory macrophages in obese adipose tissue, which are known to promote insulin resistance. How T cells are recruited to adipose tissue and activated in obesity is a topic of considerable interest. Conversely, T cell number is decreased in malnourished individuals, and T cells in the setting of malnutrition have decreased effector function and proliferative capacity. The adipokine leptin, which is secreted in proportion to adipocyte mass, may have a key role in mediating adipocyte-T cell interactions in both obesity and malnutrition, and has been shown to promote effector T cell function and metabolism while inhibiting regulatory T cell proliferation. Additionally, key molecular signals are involved in T cell metabolic adaptation during nutrient stress; among them, the metabolic regulator AMP kinase (AMPK and the mammalian target of rapamycin (mTOR have critical roles in regulating T cell number, function, and metabolism. In summary, understanding how T cell number and function are altered in obesity and malnutrition will lead to better understanding of and treatment for diseases where nutritional status determines clinical outcome.

  7. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    Science.gov (United States)

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  8. Systems biology analysis reveals role of MDM2 in diabetic nephropathy

    Science.gov (United States)

    Saito, Rintaro; Rocanin-Arjo, Anaïs; You, Young-Hyun; Darshi, Manjula; Van Espen, Benjamin; Miyamoto, Satoshi; Pham, Jessica; Pu, Minya; Romoli, Simone; Natarajan, Loki; Ju, Wenjun; Kretzler, Matthias; Nelson, Robert; Ono, Keiichiro; Thomasova, Dana; Mulay, Shrikant R.; Ideker, Trey; D’Agati, Vivette; Beyret, Ergin; Belmonte, Juan Carlos Izpisua; Anders, Hans Joachim

    2016-01-01

    To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers. PMID:27777973

  9. Revealing the role of catechol moieties in the interactions between peptides and inorganic surfaces.

    Science.gov (United States)

    Das, Priyadip; Reches, Meital

    2016-08-18

    Catechol (1,2-dihydroxy benzene) moieties are being widely used today in new adhesive technologies. Understanding their mechanism of action is therefore of high importance for developing their applications in materials science. This paper describes a single-molecule study of the interactions between catechol-related amino acid residues and a well-defined titanium dioxide (TiO2) surface. It is the first quantified measurement of the adhesion of these residues with a well-defined TiO2 surface. Single-molecule force spectroscopy measurements with AFM determined the role of different substitutions of the catechol moiety on the aromatic ring in the adhesion to the surface. These results shed light on the nature of interactions between these residues and inorganic metal oxide surfaces. This information is important for the design and fabrication of catechol-based materials such as hydrogels, coatings, and composites. Specifically, the interaction with TiO2 is important for the development of solar cells.

  10. Localization of Cladosporium fulvum hydrophobins reveals a role for HCf-6 in adhesion.

    Science.gov (United States)

    Lacroix, Hélène; Whiteford, James R; Spanu, Pietro D

    2008-09-01

    Hydrophobins are amphipathic molecules which form part of fungal cell walls and extracellular matrices and perform a variety of roles in fungal growth and development. The tomato pathogen Cladosporium fulvum has six hydrophobin genes, HCf-1 to -6. We have devised an epitope tagging approach for establishing hydrophobin localization during growth in culture and in plants. In this paper we localize HCf-2, -3, -4 and -5 and compare the data to our previous observations for HCf-1 and -6. In culture, HCf-1, -2, -3 and 4 localize to conidia and also appear on aerial hyphae. HCf-4 is unique in that it appears on submerged hyphae. HCf-5 expression is tightly regulated and appears on aerial hyphae early on during growth. Only HCf-1, -3 and -6 were observed during infection; HCf-3 appears on both conidia and emerging germ tubes. We also show that HCf-6 is secreted and coats surfaces under and around growing hyphae and demonstrate the effect of deleting HCf-6 on the adhesion of germinating C. fulvum conidia to glass slides.

  11. Long-term reduction of T-cell intracellular antigens reveals a transcriptome associated with extracellular matrix and cell adhesion components.

    Directory of Open Access Journals (Sweden)

    Mario Núñez

    Full Text Available Knockdown of T-cell intracellular antigens TIA1 and TIAR contributes to a cellular phenotype characterised by uncontrolled proliferation and tumorigenesis. Massive-scale poly(A+ RNA sequencing of TIA1 or TIAR-knocked down HeLa cells reveals transcriptome signatures comprising genes and functional categories potentially able to modulate several aspects of membrane dynamics associated with extracellular matrix and focal/cell adhesion events. The transcriptomic heterogeneity is the result of differentially expressed genes and RNA isoforms generated by alternative splicing and/or promoter usage. These results suggest a role for TIA proteins in the regulation and/or modulation of cellular homeostasis related to focal/cell adhesion, extracellular matrix and membrane and cytoskeleton dynamics.

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

    Science.gov (United States)

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

    2016-06-01

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

  13. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells

    Science.gov (United States)

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-01

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  14. A Trans-omics Mathematical Analysis Reveals Novel Functions of the Ornithine Metabolic Pathway in Cancer Stem Cells.

    Science.gov (United States)

    Koseki, Jun; Matsui, Hidetoshi; Konno, Masamitsu; Nishida, Naohiro; Kawamoto, Koichi; Kano, Yoshihiro; Mori, Masaki; Doki, Yuichiro; Ishii, Hideshi

    2016-02-11

    Bioinformatics and computational modelling are expected to offer innovative approaches in human medical science. In the present study, we performed computational analyses and made predictions using transcriptome and metabolome datasets obtained from fluorescence-based visualisations of chemotherapy-resistant cancer stem cells (CSCs) in the human oesophagus. This approach revealed an uncharacterized role for the ornithine metabolic pathway in the survival of chemotherapy-resistant CSCs. The present study fastens this rationale for further characterisation that may lead to the discovery of innovative drugs against robust CSCs.

  15. Physiological and pathological role of local and immigrating colonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Ferenc Sipos; Gábor Valcz; Béla Molnár

    2012-01-01

    The latest avenue of research is revealing the existence of and role for the colonic stem cells in the physiological renewal of the mucosa and in pathological circumstances where they have both positive and negative effects. In the case of human colon, different levels of stem cell compartments exist. First, the crypt epithelial stem cells, which have a role in the normal crypt epithelial cell dynamics and in colorectal carcinogenesis. Close to the crypts, the second layer of stem cells can be found; the local subepithelial stem cell niche, including the pericryptic subepithelial myofibroblasts that regulate the epithelial cell differentiation and have a crucial role in cancer progression and chronic inflammation-related fibrosis. The third level of stem cell compartment is the immigrating bone-marrow-derived stem cells, which have an important role in wound healing after severe mucosal inflammation, but are also involved in cancer invasion. This paper focuses on stem cell biology in the context of physiological and pathological processes in the human colon.

  16. Role of microRNAs in maintaining cancer stem cells.

    Science.gov (United States)

    Garofalo, Michela; Croce, Carlo M

    2015-01-01

    Increasing evidence sustains that the establishment and maintenance of many, if not all, human cancers are due to cancer stem cells (CSCs), tumor cells with stem cell properties, such as the capacity to self-renew or generate progenitor and differentiated cells. CSCs seem to play a major role in tumor metastasis and drug resistance, but albeit the potential clinical importance, their regulation at the molecular level is not clear. Recent studies have highlighted several miRNAs to be differentially expressed in normal and cancer stem cells and established their role in targeting genes and pathways supporting cancer stemness properties. This review focuses on the last advances on the role of microRNAs in the regulation of stem cell properties and cancer stem cells in different tumors.

  17. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

  18. Fear extinction and acute stress reactivity reveal a role of LPA(1) receptor in regulating emotional-like behaviors.

    Science.gov (United States)

    Pedraza, C; Sánchez-López, J; Castilla-Ortega, E; Rosell-Valle, C; Zambrana-Infantes, E; García-Fernández, M; Rodriguez de Fonseca, F; Chun, J; Santín, L J; Estivill-Torrús, G

    2014-09-01

    LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1-6) through which lysophosphatidic acid acts as an intercellular signaling molecule. It has been proposed that this receptor has a role in controlling anxiety-like behaviors and in the detrimental consequences of stress. Here, we sought to establish the involvement of the LPA1 receptor in emotional regulation. To this end, we examined fear extinction in LPA1-null mice, wild-type and LPA1 antagonist-treated animals. In LPA1-null mice we also characterized the morphology and GABAergic properties of the amygdala and the medial prefrontal cortex. Furthermore, the expression of c-Fos protein in the amygdala and the medial prefrontal cortex, and the corticosterone response following acute stress were examined in both genotypes. Our data indicated that the absence of the LPA1 receptor significantly inhibited fear extinction. Treatment of wild-type mice with the LPA1 antagonist Ki16425 mimicked the behavioral phenotype of LPA1-null mice, revealing that the LPA1 receptor was involved in extinction. Immunohistochemistry studies revealed a reduction in the number of neurons, GABA+ cells, calcium-binding proteins and the volume of the amygdala in LPA1-null mice. Following acute stress, LPA1-null mice showed increased corticosterone and c-Fos expression in the amygdala. In conclusion, LPA1 receptor is involved in emotional behaviors and in the anatomical integrity of the corticolimbic circuit, the deregulation of which may be a susceptibility factor for anxiety disorders and a potential therapeutic target for the treatment of these diseases.

  19. Circadian gating of the cell cycle revealed in single cyanobacterial cells.

    Science.gov (United States)

    Yang, Qiong; Pando, Bernardo F; Dong, Guogang; Golden, Susan S; van Oudenaarden, Alexander

    2010-03-19

    Although major progress has been made in uncovering the machinery that underlies individual biological clocks, much less is known about how multiple clocks coordinate their oscillations. We simultaneously tracked cell division events and circadian phases of individual cells of the cyanobacterium Synechococcus elongatus and fit the data to a model to determine when cell cycle progression slows as a function of circadian and cell cycle phases. We infer that cell cycle progression in cyanobacteria slows during a specific circadian interval but is uniform across cell cycle phases. Our model is applicable to the quantification of the coupling between biological oscillators in other organisms.

  20. Isolation of Chromatin from Dysfunctional Telomeres Reveals an Important Role for Ring1b in NHEJ-Mediated Chromosome Fusions

    Directory of Open Access Journals (Sweden)

    Cristina Bartocci

    2014-05-01

    Full Text Available When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation.

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

    DEFF Research Database (Denmark)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

    2016-01-01

    as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...

  2. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    Science.gov (United States)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  3. Role of prolactin in B cell regulation in multiple sclerosis.

    Science.gov (United States)

    Correale, Jorge; Farez, Mauricio F; Ysrraelit, María Célica

    2014-04-15

    The role of prolactin in MS pathogenesis was investigated. Prolactin levels were higher in MS subjects both during remission and exacerbation compared to control subjects. Prolactin increased JAK2 expression and Stat phosphorylation on B cells, up-regulated anti-MOG antibody secreting cell numbers, BAFF levels, and Bcl-2expression, and down-regulated expression of Trp63. Prolactin levels correlated positively with anti-MOG secreting cell numbers, and negatively with induced apoptotic B cells. Additionally, prolactin decreased B cell receptor-mediated activation threshold, and induced CD40 expression in B cells. These findings suggest that prolactin promotes B cell autoreactivity in MS through different mechanisms.

  4. Balanced transcription of cell division genes in Bacillus subtilis as revealed by single cell analysis

    NARCIS (Netherlands)

    Trip, Erik Nico; Veening, Jan-Willem; Stewart, Eric J.; Errington, Jeff; Scheffers, Dirk-Jan

    2013-01-01

    Cell division in bacteria is carried out by a set of conserved proteins that all have to function at the correct place and time. A cell cycle-dependent transcriptional programme drives cell division in bacteria such as Caulobacter crescentus. Whether such a programme exists in the Gram-positive mode

  5. Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells

    Directory of Open Access Journals (Sweden)

    Jorge E. Ramirez

    2016-12-01

    Full Text Available The brain’s control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca2+ imaging is a faithful reporter of Na+-dependent “simple spike” pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABAARs abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (inactivity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei.

  6. Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase.

    Science.gov (United States)

    Ortiz, Jorge G Muñiz; Opoka, Robert; Kane, Daniel; Cartwright, Iain L

    2009-02-01

    Chronic exposure to arsenic-contaminated drinking water can lead to a variety of serious pathological outcomes. However, differential responsiveness within human populations suggests that interindividual genetic variation plays an important role. We are using Drosophila to study toxic metal response pathways because of unrivalled access to varied genetic approaches and significant demonstrable overlap with many aspects of mammalian physiology and disease phenotypes. Genetic analysis (via chromosomal segregation and microsatellite marker-based recombination) of various wild-type strains exhibiting relative susceptibility or tolerance to the lethal toxic effects of arsenite identified a limited X-chromosomal region (16D-F) able to confer a differential response phenotype. Using an FRT-based recombination approach, we created lines harboring small, overlapping deficiencies within this region and found that relative arsenite sensitivity arose when the dose of the glutathione synthetase (GS) gene (located at 16F1) was reduced by half. Knockdown of GS expression by RNA interference (RNAi) in cultured S2 cells led to enhanced arsenite sensitivity, while GS RNAi applied to intact organisms dramatically reduced the concentration of food-borne arsenite compatible with successful growth and development. Our analyses, initially guided by observations on naturally occurring variants, provide genetic proof that an optimally functioning two-step glutathione (GSH) biosynthetic pathway is required in vivo for a robust defense against arsenite; the enzymatic implications of this are discussed in the context of GSH supply and demand under arsenite-induced stress. Given an identical pathway for human GSH biosynthesis, we suggest that polymorphisms in GSH biosynthetic genes may be an important contributor to differential arsenic sensitivity and exposure risk in human populations.

  7. Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

    Science.gov (United States)

    Hall, Hardy C; Cheung, Jingling; Ellis, Brian E

    2013-04-01

    The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

  8. Epidermal Patterning Genes Impose Non-cell Autonomous Cell Size Determination and have Additional Roles in Root Meristem Size Control

    Institute of Scientific and Technical Information of China (English)

    Christian L?fke; Kai Dünser; Jürgen Kleine-Vehn

    2013-01-01

    The regulation of cellular growth is of vital importance for embryonic and postembryonic patterning. Growth regulation in the epidermis has importance for organ growth rates in roots and shoots, proposing epidermal cells as an interesting model for cellular growth regulation. Here we assessed whether the root epidermis is a suitable model system to address cell size determination. In Arabidopsis thaliana L., root epidermal cells are regularly spaced in neighbouring tricho-(root hair) and atrichoblast (non-hair) cells, showing already distinct cell size regulation in the root meristem. We determined cell sizes in the root meristem and at the onset of cellular elongation, revealing that not only division rates but also cellular shape is distinct in tricho-and atrichoblasts. Intriguingly, epidermal-patterning mutants, failing to define differential vacuolization in neighbouring epidermal cell files, also display non-differential growth. Using these epidermal-patterning mutants, we show that polarized growth behaviour of epidermal tricho-and atrichoblast is interdependent, suggesting non-cell autonomous signals to integrate tissue expansion. Besides the interweaved cell-type-dependent growth mechanism, we reveal an additional role for epidermal patterning genes in root meristem size and organ growth regulation. We conclude that epidermal cells represent a suitable model system to study cell size determination and interdependent tissue growth.

  9. In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells.

    Science.gov (United States)

    Beckervordersandforth, Ruth; Tripathi, Pratibha; Ninkovic, Jovica; Bayam, Efil; Lepier, Alexandra; Stempfhuber, Barbara; Kirchhoff, Frank; Hirrlinger, Johannes; Haslinger, Anja; Lie, D Chichung; Beckers, Johannes; Yoder, Bradley; Irmler, Martin; Götz, Magdalena

    2010-12-03

    Until now, limitations in the ability to enrich adult NSCs (aNSCs) have hampered meaningful analysis of these cells at the transcriptome level. Here we show via a split-Cre technology that coincident activity of the hGFAP and prominin1 promoters is a hallmark of aNSCs in vivo. Sorting of cells from the adult mouse subependymal zone (SEZ) based on their expression of GFAP and prominin1 isolates all self-renewing, multipotent stem cells at high purity. Comparison of the transcriptome of these purified aNSCs to parenchymal nonneurogenic astrocytes and other SEZ cells reveals aNSC hallmarks, including neuronal lineage priming and the importance of cilia- and Ca-dependent signaling pathways. Inducible deletion of the ciliary protein IFT88 in aNSCs validates the role of ciliary function in aNSCs. Our work reveals candidate molecular regulators for unique features of aNSCs and facilitates future selective analysis of aNSCs in other functional contexts, such as aging and injury.

  10. Binding of the Galanthus nivalis agglutinin to thymocytes reveals alterations in surface glycosylation during T-cell development.

    Science.gov (United States)

    Sinkora, J; Kolínská, J; Reháková, Z; Cerný, J; Doubravská, L

    2002-02-01

    Surface binding of the Galanthus nivalis agglutinin (GNA) to thymocyte subsets has been studied in pigs and rodents by multicolour flow cytometry. In all the species examined, analogous staining profiles have been recorded. Counter-staining with anti-CD3epsilon, anti-CD4 and anti-CD8 monoclonal antibodies (MoAb) revealed that a significant increase of the GNA targets on the cell surface occurred during early thymocyte differentiation and reached its maximum at the level of the CD3loCD4+CD8+ small cortical thymocyte. This was followed by a decrease in the GNA binding capacity upon terminal maturation to the single positive thymocytes. PAGE analysis has revealed a dominant GNA-binding glycoprotein (molar mass approx. 90 kDa) present on thymocyte plasma membranes and absent on the surface of splenic lymphocytes, although both the whole cell lysates from both organs contained GNA ligands of the same size. Our findings are in agreement with previous data showing that immature thymocytes differ from their mature counterparts and peripheral T lymphocytes in the surface glycosylation pattern, and support the hypothesis that lectin-glycoprotein interaction plays a significant role in the cell-to-cell crosstalk in the thymic cortex.

  11. Computational models reveal a passive mechanism for cell migration in the crypt.

    Directory of Open Access Journals (Sweden)

    Sara-Jane Dunn

    Full Text Available Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt.

  12. Compartmental genomics in living cells revealed by single-cell nanobiopsy.

    Science.gov (United States)

    Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

    2014-01-28

    The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

  13. Giant Cell Tumor: Role of Conservative Treatment

    Institute of Scientific and Technical Information of China (English)

    Anatolii Diedkov[1; Pavlo Kovalchuk[1; Marija Kukushkina[2; Sergey Bojchuk[1; Viktor Kostyuk[1

    2014-01-01

    Giant cell tumor is aggressive bone tumor. Surgical treatment is considered to be the only effective method of treatment ofthese tumors. The problem of inoperable patients with giant cell tumors is a challenge. A total of 8 patients had giant cell bone tumorsof pelvis and sacrum. 3 patients were treated by bisphosphonates, radiation therapy and embolization of tumor-nutrient arteries. 5patients received denosumab. The efficiency was assessed according to clinical data and CT scan control. Median follow up is 28months. All 8 patients had reduction of pain intensity. Treatment with denosumab demonstrated more than 30% tumor regression. Allof the patients are in remission.

  14. Protective role of Th17 cells in pulmonary infection.

    Science.gov (United States)

    Rathore, Jitendra Singh; Wang, Yan

    2016-03-18

    Th17 cells are characterized as preferential producer of interleukins including IL-17A, IL-17F, IL-21 and IL-22. Corresponding receptors of these cytokines are expressed on number of cell types found in the mucosa, including epithelial cells and fibroblasts which constitute the prime targets of the Th17-associated cytokines. Binding of IL-17 family members to their corresponding receptors lead to modulation of antimicrobial functions of target cells including alveolar epithelial cells. Stimulated alveolar epithelial cells produce antimicrobial peptides and are involved in granulepoesis, neutrophil recruitment and tissue repair. Mucosal immunity mediated by Th17 cells is protective against numerous pulmonary pathogens including extracellular bacterial and fungal pathogens. This review focuses on the protective role of Th17 cells during pulmonary infection, highlighting subset differentiation, effector cytokines production, followed by study of the binding of these cytokines to their corresponding receptors, the subsequent signaling pathway they engender and their effector role in host defense.

  15. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

    2016-08-01

    The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  16. Role of p53 Mammary Epithelial Cell Senescence

    Science.gov (United States)

    2005-05-01

    AD Award Number: DAMD17-02-1-0509 TITLE: Role of p53 Mammary Epithelial Cell Senescence PRINCIPAL INVESTIGATOR: Goberdhan P. Dimri, Ph.D. CONTRACTING ...type and However, Mucl , K-18, and ASMA were not expressed in luminal cell type groups [12,68]. Interestingly, a significant cells present in...13,17,27], the has also attracted a great interest in the field of breast cancer candidate mammary stem cells appear to be ESA+, Mucl -, research, and

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-08

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

  18. Role of bentonite clays on cell growth.

    Science.gov (United States)

    Cervini-Silva, Javiera; Ramírez-Apan, María Teresa; Kaufhold, Stephan; Ufer, Kristian; Palacios, Eduardo; Montoya, Ascención

    2016-04-01

    Bentonites, naturally occurring clays, are produced industrially because of their adsorbent capacity but little is known about their effects on human health. This manuscript reports on the effect of bentonites on cell growth behaviour. Bentonites collected from India (Bent-India), Hungary (Bent-Hungary), Argentina (Bent-Argentina), and Indonesia (Bent-Indonesia) were studied. All four bentonites were screened in-vitro against two human cancer cell lines [U251 (central nervous system, glioblastoma) and SKLU-1 (lung adenocarcinoma)] supplied by the National Cancer Institute (USA). Bentonites induced growth inhibition in the presence of U251 cells, and growth increment in the presence of SKLU-1 cells, showing that interactions between bentonite and cell surfaces were highly specific. The proliferation response for U251 cells was explained because clay surfaces controlled the levels of metabolic growth components, thereby inhibiting the development of high-grade gliomas, particularly primary glioblastomas. On the other hand, the proliferation response for SKLU-1 was explained by an exacerbated growth favoured by swelling, and concomitant accumulation of solutes, and their hydration and transformation via clay-surface mediated reactions.

  19. Visualization of co-localization in Aβ42-administered neuroblastoma cells reveals lysosome damage and autophagosome accumulation related to cell death.

    Science.gov (United States)

    Soura, Violetta; Stewart-Parker, Maris; Williams, Thomas L; Ratnayaka, Arjuna; Atherton, Joe; Gorringe, Kirsti; Tuffin, Jack; Darwent, Elisabeth; Rambaran, Roma; Klein, William; Lacor, Pascale; Staras, Kevin; Thorpe, Julian; Serpell, Louise C

    2012-01-15

    Aβ42 [amyloid-β peptide-(1-42)] plays a central role in Alzheimer's disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. In the present study we show that administration of freshly prepared Aβ42 oligomers to a neuroblastoma (SH-SY5Y) cell line results in a reduction in survival, and that Aβ42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Aβ42 with time through the endosomal system and shows that it accumulates in lysosomes. A 24 h incubation with Aβ results in cells that have damaged lysosomes showing signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Aβ is evident in the cells and the results of the present study suggest that the addition of Aβ oligomers disrupts a crucial balance in Aβ conformation and concentration inside neuronal cells, resulting in catastrophic effects on cellular function and, ultimately, in cell death.

  20. Traumatic brain injury reveals novel cell lineage relationships within the subventricular zone

    Directory of Open Access Journals (Sweden)

    Gretchen M. Thomsen

    2014-07-01

    Full Text Available The acute response of the rodent subventricular zone (SVZ to traumatic brain injury (TBI involves a physical expansion through increased cell proliferation. However, the cellular underpinnings of these changes are not well understood. Our analyses have revealed that there are two distinct transit-amplifying cell populations that respond in opposite ways to injury. Mash1+ transit-amplifying cells are the primary SVZ cell type that is stimulated to divide following TBI. In contrast, the EGFR+ population, which has been considered to be a functionally equivalent progenitor population to Mash1+ cells in the uninjured brain, becomes significantly less proliferative after injury. Although normally quiescent GFAP+ stem cells are stimulated to divide in SVZ ablation models, we found that the GFAP+ stem cells do not divide more after TBI. We found, instead, that TBI results in increased numbers of GFAP+/EGFR+ stem cells via non-proliferative means—potentially through the dedifferentiation of progenitor cells. EGFR+ progenitors from injured brains only were competent to revert to a stem cell state following brief exposure to growth factors. Thus, our results demonstrate previously unknown changes in lineage relationships that differ from conventional models and likely reflect an adaptive response of the SVZ to maintain endogenous brain repair after TBI.

  1. Tumor-stroma interaction: Revealing fibroblast-secreted exosomes as potent regulators of Wnt-planar cell polarity signaling in cancer metastasis.

    Science.gov (United States)

    Luga, Valbona; Wrana, Jeffrey L

    2013-12-01

    Cancer-associated fibroblasts (CAF) regulate tumor progression, but their role in cancer metastasis remains largely unexplored. Exosomes are secreted microvesicles that are emerging as potent mediators of cell-cell communication that are of particular importance in tumor-stroma interactions. The Wnt-planar cell polarity (PCP) pathway is the primary regulator of convergent extension cell movements during vertebrate development, but the role of this signaling pathway in cancer cell migration and metastasis has been unclear. Recently, we revealed that fibroblasts secrete exosomes that promote breast cancer cell (BCC) protrusive activity, motility, and metastasis by activating autocrine Wnt-PCP signaling in BCCs. Moreover, we showed that Wnt ligands produced by BCCs tether to fibroblast exosomes upon trafficking of exosomes in BCCs. These findings have several implications that motivate promising future research in the fields of tumor-stroma communication, exosome function, and Wnt-PCP signaling in cancer metastasis.

  2. Programmed cell death and its role in inflammation

    Institute of Scientific and Technical Information of China (English)

    Yong Yang; Ge-Ning Jiang; Peng Zhang; Jie Fan

    2015-01-01

    Cell death plays an important role in the regulation of inflammation and may be the result of inflammation. The maintenance of tissue homeostasis necessitates both the recognition and removal of invading microbial pathogens as well as the clearance of dying cells. In the past few decades, emerging knowledge on cell death and inflammation has enriched our molecular understanding of the signaling pathways that mediate various programs of cell death and multiple types of inflammatory responses. This review provides an overview of the major types of cell death related to inflammation. Modification of cell death pathways is likely to be a logical therapeutic target for inflammatory diseases.

  3. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    Science.gov (United States)

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  4. The role of Th17 cells in anti-cancer immunity

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    Iwona Hus

    2010-05-01

    Full Text Available Th17 cells are a newly identified population of CD4 Th cells characterized by interleukin 17 production and are therefore named “Th17”. Interleukin 17 (IL-17; IL-17A is one of the key cytokines for the activation, recruitment, and migration of neutrophils, so Th17 cells have been suggested to play an important role in responses against extracellular bacteria and fungi, in which granulocyte infiltration is highly protective. Among the other cytokines secreted by Th17 cells are IL-22, IL-26, IL-6, and TNF-α. Th17 cells are considered to be involved in the pathogenesis of various inflammatory, autoimmune, and allergic diseases. Th17 cells were found in both mouse and human tumors; however, their role in the tumor microenvironment is still poorly understood IL-17, as a pleiotropic cytokine, may suppress or promote tumor growth since it was shown that it stimulated tumor growth by inducing tumor vascularization or enhancing inflammation, but some other studies revealed also opposite roles for Th17 cells in human tumors. It seems that Th17 cells may play distinct roles in cancer depending of tumor immunogenicity, the stage of development, and the impact of inflammation and angiogenesis on tumor pathogenesis.

  5. Role of Innate Lymphoid Cells in Lung Disease

    Directory of Open Access Journals (Sweden)

    SayedMehran Marashian

    2015-10-01

    Full Text Available  Innate lymphoid cells (ILCs are identified as novel population of hematopoietic cells which protect the body by coordinating the innate immune response against a wide range of threats including infections, tissue damages and homeostatic disturbances. ILCs, particularly ILC2 cells, are found throughout the body including the brain. ILCs are morphologically similar to lymphocytes, express and release high levels of T-helper (Th1, Th2 and Th17 cytokines but do not express classical cell-surface markers that are associated with other immune cell lineages.Three types of ILCs (ILC1, 2 & 3 have been reported depending upon the cytokines produced. ILC1 cells encompass natural killer (NK cells and interferon (IFN-g releasing cells; ILC2 cells release the Th2 cytokines, IL-5, IL-9 and IL-13 in response to IL-25 and IL-33; and ILC3 cells which release IL-17 and IL-22. ILC2 cells have been implicated inmucosal reactions occurring in animal models of allergic asthma and virus-induced lung disorders resulting in the regulation of airway remodeling and tissue homeostasis.There is evidence for increased ILC2 cell numbers in allergic responses in man but little is known about the role of ILCs in chronic obstructive pulmonary disease (COPD. Further understanding of the characteristics of ILCs such as their origin, location and phenotypes and function would help to clarify the role of these cells in the pathogenesis of various lung diseases.In this review we will focus on the role of ILC2 cells and consider their origin, function,location and possible role in the pathogenesis of the chronic inflammatory disorders such as asthma and COPD.   

  6. Power-Law Modeling of Cancer Cell Fates Driven by Signaling Data to Reveal Drug Effects

    Science.gov (United States)

    Zhang, Fan; Wu, Min; Kwoh, Chee Keong; Zheng, Jie

    2016-01-01

    Extracellular signals are captured and transmitted by signaling proteins inside a cell. An important type of cellular responses to the signals is the cell fate decision, e.g., apoptosis. However, the underlying mechanisms of cell fate regulation are still unclear, thus comprehensive and detailed kinetic models are not yet available. Alternatively, data-driven models are promising to bridge signaling data with the phenotypic measurements of cell fates. The traditional linear model for data-driven modeling of signaling pathways has its limitations because it assumes that the a cell fate is proportional to the activities of signaling proteins, which is unlikely in the complex biological systems. Therefore, we propose a power-law model to relate the activities of all the measured signaling proteins to the probabilities of cell fates. In our experiments, we compared our nonlinear power-law model with the linear model on three cancer datasets with phosphoproteomics and cell fate measurements, which demonstrated that the nonlinear model has superior performance on cell fates prediction. By in silico simulation of virtual protein knock-down, the proposed model is able to reveal drug effects which can complement traditional approaches such as binding affinity analysis. Moreover, our model is able to capture cell line specific information to distinguish one cell line from another in cell fate prediction. Our results show that the power-law data-driven model is able to perform better in cell fate prediction and provide more insights into the signaling pathways for cancer cell fates than the linear model. PMID:27764199

  7. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

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    Arnauld eSergé

    2016-05-01

    Full Text Available The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation and metastasis.

  8. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    Science.gov (United States)

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

  9. Computational Analysis of Transcriptional Circuitries in Human Embryonic Stem Cells Reveals Multiple and Independent Networks

    Directory of Open Access Journals (Sweden)

    Xiaosheng Wang

    2014-01-01

    Full Text Available It has been known that three core transcription factors (TFs, NANOG, OCT4, and SOX2, collaborate to form a transcriptional circuitry to regulate pluripotency and self-renewal of human embryonic stem (ES cells. Similarly, MYC also plays an important role in regulating pluripotency and self-renewal of human ES cells. However, the precise mechanism by which the transcriptional regulatory networks control the activity of ES cells remains unclear. In this study, we reanalyzed an extended core network, which includes the set of genes that are cobound by the three core TFs and additional TFs that also bind to these cobound genes. Our results show that beyond the core transcriptional network, additional transcriptional networks are potentially important in the regulation of the fate of human ES cells. Several gene families that encode TFs play a key role in the transcriptional circuitry of ES cells. We also demonstrate that MYC acts independently of the core module in the regulation of the fate of human ES cells, consistent with the established argument. We find that TP53 is a key connecting molecule between the core-centered and MYC-centered modules. This study provides additional insights into the underlying regulatory mechanisms involved in the fate determination of human ES cells.

  10. Single-Cell Transcript Profiles Reveal Multilineage Priming in Early Progenitors Derived from Lgr5+ Intestinal Stem Cells

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    Tae-Hee Kim

    2016-08-01

    Full Text Available Lgr5+ intestinal stem cells (ISCs drive epithelial self-renewal, and their immediate progeny—intestinal bipotential progenitors—produce absorptive and secretory lineages via lateral inhibition. To define features of early transit from the ISC compartment, we used a microfluidics approach to measure selected stem- and lineage-specific transcripts in single Lgr5+ cells. We identified two distinct cell populations, one that expresses known ISC markers and a second, abundant population that simultaneously expresses markers of stem and mature absorptive and secretory cells. Single-molecule mRNA in situ hybridization and immunofluorescence verified expression of lineage-restricted genes in a subset of Lgr5+ cells in vivo. Transcriptional network analysis revealed that one group of Lgr5+ cells arises from the other and displays characteristics expected of bipotential progenitors, including activation of Notch ligand and cell-cycle-inhibitor genes. These findings define the earliest steps in ISC differentiation and reveal multilineage gene priming as a fundamental property of the process.

  11. Mechanistic insights into the distribution of carbohydrate clusters on cell membranes revealed by dSTORM imaging

    Science.gov (United States)

    Chen, Junling; Gao, Jing; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

    2016-07-01

    Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the carbohydrate-binding proteins (i.e., galectins) cross-linking their specific carbohydrate ligands. Our results clarify the organizational mechanism of carbohydrates on cell surfaces from their formation, stable existence and size-restriction, which promotes a better understanding of the relationship between the function and distribution of carbohydrates, as well as the structure of cell membranes.Cell surface carbohydrates play significant roles in many physiological processes and act as primary markers to indicate various cellular physiological states. The functions of carbohydrates are always associated with their expression and distribution on cell membranes. Based on our previous work, we found that carbohydrates tend to form clusters; however, the underlying mechanism of these clusters remains unknown. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we found that with the contributions of lipid raft as a stable factor and actin cytoskeleton as a restrictive factor, carbohydrate clusters can stably exist with restricted size. Additionally, we revealed that the formation of most carbohydrate clusters (Gal and GlcANc clusters) depended on the

  12. The Role of microRNAs in Animal Cell Reprogramming.

    Science.gov (United States)

    Cruz-Santos, María Concepción; Aragón-Raygoza, Alejandro; Espinal-Centeno, Annie; Arteaga-Vázquez, Mario; Cruz-Hernández, Andrés; Bako, Laszlo; Cruz-Ramírez, Alfredo

    2016-07-15

    Our concept of cell reprogramming and cell plasticity has evolved since John Gurdon transferred the nucleus of a completely differentiated cell into an enucleated Xenopus laevis egg, thereby generating embryos that developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4, and c-myc has evidenced the plasticity of the genome to change the expression program and cell phenotype by driving differentiated cells to the pluripotent state. Beyond these milestone achievements, research in artificial cell reprogramming has been focused on other molecules that are different than transcription factors. Among the candidate molecules, microRNAs (miRNAs) stand out due to their potential to control the levels of proteins that are involved in cellular processes such as self-renewal, proliferation, and differentiation. Here, we review the role of miRNAs in the maintenance and differentiation of mesenchymal stem cells, epimorphic regeneration, and somatic cell reprogramming to induced pluripotent stem cells.

  13. Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.

    Science.gov (United States)

    Grover, Amit; Sanjuan-Pla, Alejandra; Thongjuea, Supat; Carrelha, Joana; Giustacchini, Alice; Gambardella, Adriana; Macaulay, Iain; Mancini, Elena; Luis, Tiago C; Mead, Adam; Jacobsen, Sten Eirik W; Nerlov, Claus

    2016-03-24

    Aged haematopoietic stem cells (HSCs) generate more myeloid cells and fewer lymphoid cells compared with young HSCs, contributing to decreased adaptive immunity in aged individuals. However, it is not known how intrinsic changes to HSCs and shifts in the balance between biased HSC subsets each contribute to the altered lineage output. Here, by analysing HSC transcriptomes and HSC function at the single-cell level, we identify increased molecular platelet priming and functional platelet bias as the predominant age-dependent change to HSCs, including a significant increase in a previously unrecognized class of HSCs that exclusively produce platelets. Depletion of HSC platelet programming through loss of the FOG-1 transcription factor is accompanied by increased lymphoid output. Therefore, increased platelet bias may contribute to the age-associated decrease in lymphopoiesis.

  14. The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

    Directory of Open Access Journals (Sweden)

    Menu Eline

    2002-03-01

    Full Text Available Abstract Background Liver sinusoidal endothelial cells (LSECs react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. Results Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers. Conclusion (I A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

  15. Lassa Virus Cell Entry Reveals New Aspects of Virus-Host Cell Interaction.

    Science.gov (United States)

    Torriani, Giulia; Galan-Navarro, Clara; Kunz, Stefan

    2017-02-15

    Viral entry represents the first step of every viral infection and is a determinant for the host range and disease potential of a virus. Here, we review the latest developments on cell entry of the highly pathogenic Old World arenavirus Lassa virus, providing novel insights into the complex virus-host cell interaction of this important human pathogen. We will cover new discoveries on the molecular mechanisms of receptor recognition, endocytosis, and the use of late endosomal entry factors.

  16. Proteomics analysis of EV71-infected cells reveals the involvement of host protein NEDD4L in EV71 replication.

    Science.gov (United States)

    Kuo, Rei-Lin; Lin, Ya-Han; Wang, Robert Yung-Liang; Hsu, Chia-Wei; Chiu, Yi-Ting; Huang, Hsing-I; Kao, Li-Ting; Yu, Jau-Song; Shih, Shin-Ru; Wu, Chih-Ching

    2015-04-01

    Enterovirus 71 (EV71) is a human enterovirus that has seriously affected the Asia-Pacific area for the past two decades. EV71 infection can result in mild hand-foot-and-mouth disease and herpangina and may occasionally lead to severe neurological complications in children. However, the specific biological processes that become altered during EV71 infection remain unclear. To further explore host responses upon EV71 infection, we identified proteins differentially expressed in EV71-infected human glioblastoma SF268 cells using isobaric mass tag (iTRAQ) labeling coupled with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Network analysis of proteins altered in cells infected with EV71 revealed that the changed biological processes are related to protein and ion transport, regulation of protein degradation, and homeostatic processes. We confirmed that the levels of NEDD4L and PSMF1 were increased and reduced, respectively, in EV71-infected cells compared to mock-infected control cells. To determine the physiological relevance of our findings, we investigated the consequences of EV71 infection in cells with NEDD4L or PSMF1 depletion. We found that the depletion of NEDD4L significantly reduced the replication of EV71, whereas PSMF1 knockdown enhanced EV71 replication. Collectively, our findings provide the first evidence of proteome-wide dysregulation by EV71 infection and suggest a novel role for the host protein NEDD4L in the replication of this virus.

  17. Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

    Science.gov (United States)

    Buetti-Dinh, Antoine; O’Hare, Thomas

    2016-01-01

    A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance. PMID:27669408

  18. Role of Dendritic Cells in Immune Dysfunction

    Science.gov (United States)

    Savary, Cherylyn A.

    1997-01-01

    Specific aims include: (1) Application of the bioreactor to enhance cytokine-regulated proliferation and maturation of dendritic cells (DC); (2) Based on clues from spaceflight: compare the frequency and function of DC in normal donors and immunocompromised cancer patients; and (3) Initiate studies on the efficiency of cytokine therapy and DC-assisted immunotherapy (using bioreactor-expanded DC) in animal models of experimental fungal infections.

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

    Directory of Open Access Journals (Sweden)

    Chien-Chih Ke

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

  20. Role of cell-cell adhesion complexes in embryonic stem cell biology.

    Science.gov (United States)

    Pieters, Tim; van Roy, Frans

    2014-06-15

    Pluripotent embryonic stem cells (ESCs) can self-renew or differentiate into any cell type within an organism. Here, we focus on the roles of cadherins and catenins - their cytoplasmic scaffold proteins - in the fate, maintenance and differentiation of mammalian ESCs. E-cadherin is a master stem cell regulator that is required for both mouse ESC (mESC) maintenance and differentiation. E-cadherin interacts with key components of the naive stemness pathway and ablating it prevents stem cells from forming well-differentiated teratomas or contributing to chimeric animals. In addition, depleting E-cadherin converts naive mouse ESCs into primed epiblast-like stem cells (EpiSCs). In line with this, a mesenchymal-to-epithelial transition (MET) occurs during reprogramming of somatic cells towards induced pluripotent stem cells (iPSCs), leading to downregulation of N-cadherin and acquisition of high E-cadherin levels. β-catenin exerts a dual function; it acts in cadherin-based adhesion and in WNT signaling and, although WNT signaling is important for stemness, the adhesive function of β-catenin might be crucial for maintaining the naive state of stem cells. In addition, evidence is rising that other junctional proteins are also important in ESC biology. Thus, precisely regulated levels and activities of several junctional proteins, in particular E-cadherin, safeguard naive pluripotency and are a prerequisite for complete somatic cell reprogramming.

  1. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

    Science.gov (United States)

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-07-07

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.

  2. Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF-β responsiveness

    Science.gov (United States)

    Marinković, Aleksandar; Mih, Justin D.; Park, Jin-Ah; Liu, Fei

    2012-01-01

    Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation. PMID:22659883

  3. Systematic Perturbation of Cytoskeletal Function Reveals a Linear Scaling Relationship between Cell Geometry and Fitness

    Directory of Open Access Journals (Sweden)

    Russell D. Monds

    2014-11-01

    Full Text Available Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between “feast-or-famine” growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  4. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    Science.gov (United States)

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  5. The role of CD44 in fetal and adult hematopoietic stem cell regulation.

    Science.gov (United States)

    Cao, Huimin; Heazlewood, Shen Y; Williams, Brenda; Cardozo, Daniela; Nigro, Julie; Oteiza, Ana; Nilsson, Susan K

    2016-01-01

    Throughout development, hematopoietic stem cells migrate to specific microenvironments, where their fate is, in part, extrinsically controlled. CD44 standard as a member of the cell adhesion molecule family is extensively expressed within adult bone marrow and has been previously reported to play important roles in adult hematopoietic regulation via CD44 standard-ligand interactions. In this manuscript, CD44 expression and function are further assessed and characterized on both fetal and adult hematopoietic stem cells. Using a CD44(-/-) mouse model, conserved functional roles of CD44 are revealed throughout development. CD44 is critical in the maintenance of hematopoietic stem and progenitor pools, as well as in hematopoietic stem cell migration. CD44 expression on hematopoietic stem cells as well as other hematopoietic cells within the bone marrow microenvironment is important in the homing and lodgment of adult hematopoietic stem cells isolated from the bone/bone marrow interface. CD44 is also involved in fetal hematopoietic stem cell migration out of the liver, via a process involving stromal cell-derived factor-1α. The absence of CD44 in neonatal bone marrow has no impact on the size of the long-term reconstituting hematopoietic stem cell pool, but results in an enhanced long-term engraftment potential of hematopoietic stem cells.

  6. Pathogenic role of mast cells in experimental eosinophilic esophagitis

    OpenAIRE

    Niranjan, Rituraj; Mavi, Parm; Rayapudi, Madhavi; Dynda, Scott; Mishra, Anil

    2013-01-01

    Eosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal intraepithelial eosinophils, extracellular eosinophil granule deposition, induced mast cell accumulation, and epithelial cell hyperplasia. However, the processes involved in the development of a number of these characteristics are largely unknown. Herein, we tested the hypothesis whether induced mast cell accumulation in the esophagus has a role in promoting EoE pathogenesis. Accordingly, we induced exper...

  7. Emerging role of Notch in stem cells and cancer.

    Science.gov (United States)

    Wang, Zhiwei; Li, Yiwei; Banerjee, Sanjeev; Sarkar, Fazlul H

    2009-06-28

    The Notch signaling pathway is known to be responsible for maintaining a balance between cell proliferation and death and, as such, plays important roles in the formation of many types of human tumors. Recently, Notch signaling pathway has been shown to control stem cell self-renewal and multi-potency. As many cancers are thought to be developed from a number of cancer stem-like cells, which are also known to be linked with the acquisition of epithelial-mesenchymal transition (EMT); and thus suggesting an expanding role of Notch signaling in human tumor progression.

  8. Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

    DEFF Research Database (Denmark)

    Neve, Bernadette; Fernandez-Zapico, Martin E; Ashkenazi-Katalan, Vered

    2005-01-01

    a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.......KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first...... in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1...

  9. On The Role of Natural Killer Cells in Neurodegenerative Diseases

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    Azzam A. Maghazachi

    2013-02-01

    Full Text Available Natural killer (NK cells exert important immunoregulatory functions by releasing several inflammatory molecules, such as IFN-γ and members of chemokines, which include CCL3/MIP-1α and CCL4/MIP-1β. These cells also express heptahelical receptors, which are coupled to heterotrimeric G proteins that guide them into inflamed and injured tissues. NK cells have been shown to recognize and destroy transformed cells and virally-infected cells, but their roles in neurodegenerative diseases have not been examined in detail. In this review, I will summarize the effects of NK cells in two neurodegenerative diseases, namely multiple sclerosis and globoid cell leukodystrophy. It is hoped that the knowledge obtained from these diseases may facilitate building rational protocols for treating these and other neurodegenerative or autoimmune diseases using NK cells and drugs that activate them as therapeutic tools.

  10. Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

    Science.gov (United States)

    Looney, Timothy J.; Zhang, Li; Chen, Chih-Hsin; Lee, Jae Hyun; Chari, Sheila; Mao, Frank Fuxiang; Pelizzola, Mattia; Zhang, Lu; Lister, Ryan; Baker, Samuel W.; Fernandes, Croydon J.; Gaetz, Jedidiah; Foshay, Kara M.; Clift, Kayla L.; Zhang, Zhenyu; Li, Wei-Qiang; Vallender, Eric J.; Wagner, Ulrich; Qin, Jane Yuxia; Michelini, Katelyn J.; Bugarija, Branimir; Park, Donghyun; Aryee, Emmanuel; Stricker, Thomas; Zhou, Jie; White, Kevin P.; Ren, Bing; Schroth, Gary P.; Ecker, Joseph R.; Xiang, Andy Peng; Lahn, Bruce T.

    2014-01-01

    Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed “cis-silenced” (or “occluded”) genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease. PMID:24310002

  11. Distinct populations of innate CD8+ T cells revealed in a CXCR3 reporter mouse.

    Science.gov (United States)

    Oghumu, Steve; Dong, Ran; Varikuti, Sanjay; Shawler, Todd; Kampfrath, Thomas; Terrazas, Cesar A; Lezama-Davila, Claudio; Ahmer, Brian M M; Whitacre, Caroline C; Rajagopalan, Sanjay; Locksley, Richard; Sharpe, Arlene H; Satoskar, Abhay R

    2013-03-01

    CXCR3, expressed mainly on activated T and NK cells, is implicated in a host of immunological conditions and can contribute either to disease resolution or pathology. We report the generation and characterization of a novel CXCR3 internal ribosome entry site bicistronic enhanced GFP reporter (CIBER) mouse in which enhanced GFP expression correlates with surface levels of CXCR3. Using CIBER mice, we identified two distinct populations of innate CD8(+) T cells based on constitutive expression of CXCR3. We demonstrate that CXCR3(+) innate CD8(+) T cells preferentially express higher levels of Ly6C and CD122, but lower levels of CCR9 compared with CXCR3(-) innate CD8(+) T cells. Furthermore, we show that CXCR3(+) innate CD8(+) T cells express higher transcript levels of antiapoptotic but lower levels of proapoptotic factors, respond more robustly to IL-2 and IL-15, and produce significantly more IFN-γ and granzyme B. Interestingly, CXCR3(+) innate CD8(+) T cells do not respond to IL-12 or IL-18 alone, but produce significant amounts of IFN-γ on stimulation with a combination of these cytokines. Taken together, these findings demonstrate that CXCR3(+) and CXCR3(-) innate CD8(+) T cells are phenotypically and functionally distinct. These newly generated CIBER mice provide a novel tool for studying the role of CXCR3 and CXCR3-expressing cells in vivo.

  12. Role of adipose-derived stem cells in wound healing.

    Science.gov (United States)

    Hassan, Waqar Ul; Greiser, Udo; Wang, Wenxin

    2014-01-01

    Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow-derived stem cells, adipose-derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro-angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration.

  13. Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

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    Roberto Plebani

    2012-11-01

    Full Text Available mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (≈2 x 10-5 of all mRNA. Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

  14. Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript.

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    Maurice A Canham

    2010-05-01

    Full Text Available ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically "undifferentiated" cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V(+S(+, appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours

  15. Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript

    Science.gov (United States)

    Canham, Maurice A.; Sharov, Alexei A.; Ko, Minoru S. H.; Brickman, Joshua M.

    2010-01-01

    ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically “undifferentiated” cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly

  16. Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins

    Science.gov (United States)

    CRUZ*, ISA N.; COLEY*, HELEN M.; KRAMER, HOLGER B.; MADHURI, THUMULURU KAVITAH; SAFUWAN, NUR A.M.; ANGELINO, ANA RITA; YANG, MIN

    2016-01-01

    Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. Materials and Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer. *These Authors contributed equally to this study. PMID:28031236

  17. ROLE OF HEMATOPOIETIC STEM CELL TRANSPLANTATION

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    Angelo Michele Carella

    2012-10-01

    Full Text Available Hodgkin lymphoma is one of the most curable human tumors. Despite this, about 30% of these patients relapsed or are primary refractory to the first line treatment. Autografting is generally considered the standard of care for these patients. Alternative salvage strategies have been evaluated such as high dose sequential and tandem autografting strategies. In younger patients,  refractory or early relapsed after autografting, allogeneic stem cell transplantation has been employed but this approach has been followed by significant concerns since the treatment related mortality often exceeded 40-50%, and relapses were not uncommon. It is clear that patient selection remains an issue in all allografting reports. At the end, new drugs and novel treatment strategies, that are based on our understanding of the disease biology and signaling pathways, are needed to improve treatment outcome for these patients. The two leading compounds Brentuximab Vedotin and Panobinostat, are currently under evaluation  in several clinical trials.

  18. Male and female roles in crosses of Aspergillus nidulans as revealed by vegetatively incompatible parents

    NARCIS (Netherlands)

    Bruggeman, I.M.; Debets, A.J.M.; Swart, K.; Hoekstra, R.F.

    2003-01-01

    To resolve the role of male and female nuclei and mitochondria in cleistothecium formation in the model organism Aspergillus nidulans, we analysed the genetic constituents of cleistothecia, from crosses between vegetatively compatible and incompatible parents. We used markers that enabled us to dete

  19. Early transcriptional and epigenetic regulation of CD8(+) T cell differentiation revealed by single-cell RNA sequencing.

    Science.gov (United States)

    Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E; He, Zhaoren; Aigner, Stefan; Metz, Patrick J; Yu, Bingfei; Wehrens, Ellen J; Lopez, Justine; Kim, Stephanie H; Zuniga, Elina I; Goldrath, Ananda W; Chang, John T; Yeo, Gene W

    2017-04-01

    During microbial infection, responding CD8(+) T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8(+) T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8(+) T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

  20. Metabolomics reveals a role for the chromatin-binding protein HMGN5 in glutathione metabolism.

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    Eric D Ciappio

    Full Text Available High mobility group nucleosome-binding protein 5 (HMGN5 is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5(+/Y, mice with a targeted mutation in the HMGN5 gene (Hmgn5(tm1/Y, had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5(tm1/Y and their Hmgn5(+/Y littermates. Hmgn5(tm1/Y mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6 and hexokinase 1 (Hk1, was significantly decreased in Hmgn5(tm1/Y mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5(tm1/Y mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.

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

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    Ingrid R. Cordeiro

    2015-09-01

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

  2. Intracellular CHO cell metabolite profiling reveals steady-state dependent metabolic fingerprints in perfusion culture.

    Science.gov (United States)

    Karst, Daniel J; Steinhoff, Robert; Kopp, Marie R G; Serra, Elisa; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2016-12-20

    Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, Nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 10(6) cells/mL over 26 days of culture. On the other hand the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60 and 40 × 10(6) cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar and lipid precursors explained most of the variance between the different cell density set points. This article is protected by copyright. All rights reserved.

  3. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter.

    Science.gov (United States)

    Pan, Xin; Liu, Jie; Nguyen, Tiffany; Liu, Chengyu; Sun, Junhui; Teng, Yanjie; Fergusson, Maria M; Rovira, Ilsa I; Allen, Michele; Springer, Danielle A; Aponte, Angel M; Gucek, Marjan; Balaban, Robert S; Murphy, Elizabeth; Finkel, Toren

    2013-12-01

    Mitochondrial calcium has been postulated to regulate a wide range of processes from bioenergetics to cell death. Here, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. The lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death, although MCU(-/-) hearts fail to respond to the PTP inhibitor cyclosporin A. Taken together, these results clarify how acute alterations in mitochondrial matrix calcium can regulate mammalian physiology.

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

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    Kaoru Miyazaki

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

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

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    Xuewei Wang

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

  6. Dendritic cells and their role in periodontal disease.

    Science.gov (United States)

    Wilensky, A; Segev, H; Mizraji, G; Shaul, Y; Capucha, T; Shacham, M; Hovav, A-H

    2014-03-01

    T cells, particularly CD4+ T cells, play a central role in both progression and control of periodontal disease, whereas the contribution of the various CD4+ T helper subsets to periodontal destruction remains controversial, the activation, and regulation of these cells is orchestrated by dendritic cells. As sentinels of the oral mucosa, dendritic cells encounter and capture oral microbes, then migrate to the lymph node where they regulate the differentiation of CD4+ T cells. It is thus clear that dendritic cells are of major importance in the course of periodontitis, as they hold the immunological cues delivered by the pathogen and the surrounding environment, allowing them to induce destructive immunity. In recent years, advanced immunological techniques and new mouse models have facilitated in vivo studies that have provided new insights into the developmental and functional aspects of dendritic cells. This progress has also benefited the characterization of oral dendritic cells, as well as to their function in periodontitis. Here, we provide an overview of the various gingival dendritic cell subsets and their distribution, while focusing on their role in periodontal bone loss.

  7. Role of Kupffer cells in the pathogenesis of liver disease

    Institute of Scientific and Technical Information of China (English)

    George Kolios; Vassilis Valatas; Elias Kouroumalis

    2006-01-01

    Kupffer cells, the resident liver macrophages have long been considered as mostly scavenger cells responsible for removing particulate material from the portal circulation. However, evidence derived mostly from animal models, indicates that Kupffer cells may be implicated in the pathogenesis of various liver diseases including viral hepatitis, steatohepatitis, alcoholic liver disease, intrahepatic cholostasis, activation or rejection of the liver during liver transplantation and liver fibrosis. There is accumulating evidence, reviewed in this paper, suggesting that Kupffer cells may act both as effector cells in the destruction of hepatocytes by producing harmful soluble mediators as well as antigen presenting cells during viral infections of the liver. Moreover they may represent a significant source of chemoattractant molecules for cytotoxic CD8 and regulatory T cells. Their role in fibrosis is well established as they are one of the main sources of TGFβ1 production, which leads to the transformation of stellate cells into myofibroblasts. Whether all these variable functions in the liver are mediated by different Kupffer cell subpopulations remains to be evaluated. In this review we propose a model that demonstrates the role of Kupffer cells in the pathogenesis of liver disease.

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

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    Schuren Frank H

    2008-12-01

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

  9. Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation

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    Zheng Zhang

    2016-09-01

    Full Text Available Cancer genomics has provided an unprecedented opportunity for understanding genetic causes of human cancer. However, distinguishing which mutations are functionally relevant to cancer pathogenesis remains a major challenge. We describe here a mammary stem cell (MaSC organoid-based approach for rapid generation of somatic genetically engineered mouse models (GEMMs. By using RNAi and CRISPR-mediated genome engineering in MaSC-GEMMs, we have discovered that inactivation of Ptpn22 or Mll3, two genes mutated in human breast cancer, greatly accelerated PI3K-driven mammary tumorigenesis. Using these tumor models, we have also identified genetic alterations promoting tumor metastasis and causing resistance to PI3K-targeted therapy. Both Ptpn22 and Mll3 inactivation resulted in disruption of mammary gland differentiation and an increase in stem cell activity. Mechanistically, Mll3 deletion enhanced stem cell activity through activation of the HIF pathway. Thus, our study has established a robust in vivo platform for functional cancer genomics and has discovered functional breast cancer mutations.

  10. Role of autophagy in differential sensitivity of hepatocarcinoma cells to sorafenib

    Institute of Scientific and Technical Information of China (English)

    Trevan; D; Fischer; Jin-Hee; Wang; Adrian; Vlada; Jae-Sung; Kim; Kevin; E; Behrns

    2014-01-01

    AIM: To investigate the role of sorafenib(SFN) in autophagy of hepatocellular carcinoma(HCC). We evaluated how SFN affects autophagy signaling pathway in human HCC cell lines. METHODS: Two different human HCC cell lines, Hep3 B and Huh7, were subjected to different concentrations of SFN. Cell viability and onset of apoptosis were determined with colorimetric assay and immunoblotting analysis, respectively. The changes in autophagy-related proteins, including LC3, ULK1, AMPK, and LKB, were determined with immunoblotting analysis in the presence or absence of SFN. To assess autophagic dynamics, autophagic flux was measured with chloroquine, a lysosomal inhibitor. The autophagic responsiveness between different HCC cell lines was compared under the autophagy enhancing conditions.RESULTS: Hep3 B cells were significantly more resistant to SFN than Huh7 cells. Immunoblotting analysis revealed a marked increase in SFN-mediated autophagy flux in Huh7 cells, which was, however, absent in Hep3 B cells. While both starvation and rapamycin enhanced autophagy in Huh7 cells, only rapamycin increased autophagy in Hep3 B cells. Immunoblotting analysis of autophagy initiation proteins showed that SFN substantially increased phosphorylation of AMPK and consequently autophagy in Huh7, but not in Hep3 B cells.CONCLUSION: The autophagic responsiveness to SFN is distinct between Hep3 B and Huh7 cells. Resistance of Hep3 B cells to SFN may be associated with altered autophagy signaling pathways.

  11. Zebrafish yap1 plays a role in differentiation of hair cells in posterior lateral line

    Science.gov (United States)

    Loh, Siau-Lin; Teh, Cathleen; Muller, Julius; Guccione, Ernesto; Hong, Wanjin; Korzh, Vladimir

    2014-03-01

    The evolutionarily conserved Hippo signaling pathway controls organ size by regulating cell proliferation and apoptosis and this process involves Yap1. The zebrafish Yap1 acts during neural differentiation, but its function is not fully understood. The detailed analysis of yap1 expression in proliferative regions, revealed it in the otic placode that gives rise to the lateral line system affected by the morpholino-mediated knockdown of Yap1. The comparative microarray analysis of transcriptome of Yap1-deficient embryos demonstrated changes in expression of many genes, including the Wnt signaling pathway and, in particular, prox1a known for its role in development of mechanoreceptors in the lateral line. The knockdown of Yap1 causes a deficiency of differentiation of mechanoreceptors, and this defect can be rescued by prox1a mRNA. Our studies revealed a role of Yap1 in regulation of Wnt signaling pathway and its target Prox1a during differentiation of mechanosensory cells.

  12. Single cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming

    Science.gov (United States)

    Kim, Daniel H.; Marinov, Georgi K.; Pepke, Shirley; Singer, Zakary S.; He, Peng; Williams, Brian; Schroth, Gary P.; Elowitz, Michael B.; Wold, Barbara J.

    2014-01-01

    SUMMARY Cellular reprogramming highlights the epigenetic plasticity of the somatic cell state. Long noncoding RNAs (lncRNAs) have emerging roles in epigenetic regulation, but their potential functions in reprogramming cell fate have been largely unexplored. We used single-cell RNA sequencing to characterize the expression patterns of over 16,000 genes, including 437 lncRNAs, during defined stages of reprogramming to pluripotency. Self-organizing maps (SOMs) were used as an intuitive way to structure and interrogate transcriptome data at the single-cell level. Early molecular events during reprogramming involved the activation of Ras signaling pathways, along with hundreds of lncRNAs. Loss-of-function studies showed that activated lncRNAs can repress lineage-specific genes, while lncRNAs activated in multiple reprogramming cell types can regulate metabolic gene expression. Our findings demonstrate that reprogramming cells activate defined sets of functionally relevant lncRNAs and provide a resource to further investigate how dynamic changes in the transcriptome reprogram cell state. PMID:25575081

  13. Vestibular role of KCNQ4 and KCNQ5 K+ channels revealed by mouse models

    NARCIS (Netherlands)

    G. Spitzmaul (Guillermo); L. Tolosa (Leonardo); B.H.J. Winkelman (Beerend); M. Heidenreich (Matthias); M.A. Frens (Maarten); C. Chabbert (Christian); C.I. de Zeeuw (Chris); T.J. Jentsch (Thomas)

    2013-01-01

    textabstractThe function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K+ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K+ channel, which is mutated in DFNA2 human hearing loss, is expre

  14. A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles

    DEFF Research Database (Denmark)

    Wagner, Sebastian Alexander; Beli, Petra; Weinert, Brian Tate;

    2011-01-01

    Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-s...

  15. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition

    Directory of Open Access Journals (Sweden)

    Huynh Truc

    2007-12-01

    Full Text Available Abstract Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18 that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.

  16. A protective role for dengue virus-specific CD8+ T cells.

    Science.gov (United States)

    Yauch, Lauren E; Zellweger, Raphaël M; Kotturi, Maya F; Qutubuddin, Afrina; Sidney, John; Peters, Bjoern; Prestwood, Tyler R; Sette, Alessandro; Shresta, Sujan

    2009-04-15

    Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

  17. Gender Stereotypes and Gender Roles Revealed in the Students Written Work

    OpenAIRE

    Ekşi, Gonca

    2014-01-01

    Masculinity and femininity are two words that entail two opposite images with distinct sets of behaviour and personal attributes. Regardless of how well these definitions fit to each individual man and woman in society, these gender stereotypes are maintained and transmitted within the society throughout years. In return, gender stereotypes and roles affect men and women in society. In this study, 96 English Language Teaching (ELT) junior students are given a questionnaire to get socio-econom...

  18. Human genetic deficiencies reveal the roles of complement in the inflammatory network: lessons from nature

    DEFF Research Database (Denmark)

    Lappegård, Knut Tore; Christiansen, Dorte; Pharo, Anne;

    2009-01-01

    Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies--nature's own...... of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to gram-negative bacteria....

  19. Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

    Science.gov (United States)

    Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

    2017-01-01

    Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

  20. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  1. Quantitative morphometry of electrophysiologically identified CA3b interneurons reveals robust local geometry and distinct cell classes.

    Science.gov (United States)

    Ascoli, Giorgio A; Brown, Kerry M; Calixto, Eduardo; Card, J Patrick; Galván, E J; Perez-Rosello, T; Barrionuevo, Germán

    2009-08-20

    The morphological and electrophysiological diversity of inhibitory cells in hippocampal area CA3 may underlie specific computational roles and is not yet fully elucidated. In particular, interneurons with somata in strata radiatum (R) and lacunosum-moleculare (L-M) receive converging stimulation from the dentate gyrus and entorhinal cortex as well as within CA3. Although these cells express different forms of synaptic plasticity, their axonal trees and connectivity are still largely unknown. We investigated the branching and spatial patterns, plus the membrane and synaptic properties, of rat CA3b R and L-M interneurons digitally reconstructed after intracellular labeling. We found considerable variability within but no difference between the two layers, and no correlation between morphological and biophysical properties. Nevertheless, two cell types were identified based on the number of dendritic bifurcations, with significantly different anatomical and electrophysiological features. Axons generally branched an order of magnitude more than dendrites. However, interneurons on both sides of the R/L-M boundary revealed surprisingly modular axodendritic arborizations with consistently uniform local branch geometry. Both axons and dendrites followed a lamellar organization, and axons displayed a spatial preference toward the fissure. Moreover, only a small fraction of the axonal arbor extended to the outer portion of the invaded volume, and tended to return toward the proximal region. In contrast, dendritic trees demonstrated more limited but isotropic volume occupancy. These results suggest a role of predominantly local feedforward and lateral inhibitory control for both R and L-M interneurons. Such a role may be essential to balance the extensive recurrent excitation of area CA3 underlying hippocampal autoassociative memory function.

  2. Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification.

    Science.gov (United States)

    Zdravkovic, Tamara; Nazor, Kristopher L; Larocque, Nicholas; Gormley, Matthew; Donne, Matthew; Hunkapillar, Nathan; Giritharan, Gnanaratnam; Bernstein, Harold S; Wei, Grace; Hebrok, Matthias; Zeng, Xianmin; Genbacev, Olga; Mattis, Aras; McMaster, Michael T; Krtolica, Ana; Valbuena, Diana; Simón, Carlos; Laurent, Louise C; Loring, Jeanne F; Fisher, Susan J

    2015-12-01

    Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines.

  3. Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging

    Science.gov (United States)

    Shagaghi, Nadin; Bhave, Mrinal; Palombo, Enzo A.; Clayton, Andrew H. A.

    2017-01-01

    To determine the mechanism(s) of action of antimicrobial peptides (AMPs) it is desirable to provide details of their interaction kinetics with cellular, sub-cellular and molecular targets. The synthetic peptide, PuroA, displays potent antimicrobial activities which have been attributed to peptide-induced membrane destabilization, or intracellular mechanisms of action (DNA-binding) or both. We used time-lapse fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM) to directly monitor the localization and interaction kinetics of a FITC- PuroA peptide on single Candida albicans cells in real time. Our results reveal the sequence of events leading to cell death. Within 1 minute, FITC-PuroA was observed to interact with SYTO-labelled nucleic acids, resulting in a noticeable quenching in the fluorescence lifetime of the peptide label at the nucleus of yeast cells, and cell-cycle arrest. A propidium iodide (PI) influx assay confirmed that peptide translocation itself did not disrupt the cell membrane integrity; however, PI entry occurred 25–45 minutes later, which correlated with an increase in fractional fluorescence of pores and an overall loss of cell size. Our results clarify that membrane disruption appears to be the mechanism by which the C. albicans cells are killed and this occurs after FITC-PuroA translocation and binding to intracellular targets. PMID:28252014

  4. Biomaterial-enabled delivery of SDF-1α at the ventral side of breast cancer cells reveals a crosstalk between cell receptors to promote the invasive phenotype.

    Science.gov (United States)

    Liu, Xi Qiu; Fourel, Laure; Dalonneau, Fabien; Sadir, Rabia; Leal, Salome; Lortat-Jacob, Hugues; Weidenhaupt, Marianne; Albiges-Rizo, Corinne; Picart, Catherine

    2017-05-01

    The SDF-1α chemokine (CXCL12) is a potent bioactive chemoattractant known to be involved in hematopoietic stem cell homing and cancer progression. The associated SDF-1α/CXCR4 receptor signaling is a hallmark of aggressive tumors, which can metastasize to distant sites such as lymph nodes, lung and bone. Here, we engineered a biomimetic tumoral niche made of a thin and soft polyelectrolyte film that can retain SDF-1α to present it, in a spatially-controlled manner, at the ventral side of the breast cancer cells. Matrix-bound SDF-1α but not soluble SDF-1α induced a striking increase in cell spreading and migration in a serum-containing medium, which was associated with the formation of lamellipodia and filopodia in MDA-MB231 cells and specifically mediated by CXCR4. Other Knockdown and inhibition experiments revealed that CD44, the major hyaluronan receptor, acted in concert, via a spatial coincidence, to drive a specific matrix-bound SDFα-induced cell response associated with ERK signaling. In contrast, the β1 integrin adhesion receptor played only a minor role on cell polarity. The CXCR4/CD44 mediated cellular response to matrix-bound SDF-1α involved the Rac1 RhoGTPase and was sustained solely in the presence of matrix-bound SDFα, in contrast with the transient signaling observed in response to soluble SDF-1α. Our results highlight that a biomimetic tumoral niche enables to reveal potent cellular effects and so far hidden molecular mechanisms underlying the breast cancer response to chemokines. These results open new insights for the design of future innovative therapies in metastatic cancers, by inhibiting CXCR4-mediated signaling in the tumoral niche via dual targeting of receptors (CXCR4 and CD44) or of associated signaling molecules (CXCR4 and Rac1).

  5. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Schlie-Wolter, Sabrina, E-mail: s.schlie@lzh.de [Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany); Ngezahayo, Anaclet, E-mail: ngezahayo@biophysik.uni-hannover.de [Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, Hannover 30419 (Germany); Chichkov, Boris N., E-mail: b.chichkov@lzh.de [Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover (Germany)

    2013-06-10

    Cell binding to the extracellular matrix (ECM) is essential for cell and tissue functions. In this context, each tissue consists of a unique ECM composition, which may be responsible for tissue-specific cell responses. Due to the complexity of ECM-cell interactions—which depend on the interplay of inside-out and outside-in signaling cascades, cell and tissue specificity of ECM-guidance is poorly understood. In this paper, we investigate the role of different ECM components like laminin, fibronectin, and collagen type I with respect to the essential cell behaviour patterns: attachment dynamics such as adhesion kinetic and force, formation of focal adhesion complexes, morphology, proliferation, and intercellular communication. A detailed in vitro comparison of fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, and chondrocytes reveals significant differences in their cell responses to the ECM: cell behaviour follows a cell specific ligand priority ranking, which was independent of the cell type origin. Fibroblasts responded best to fibronectin, chondrocytes best to collagen I, the other cell types best to laminin. This knowledge is essential for optimization of tissue-biomaterial interfaces in all tissue engineering applications and gives insight into tissue-specific cell guidance. -- Highlights: • We analyse the impact of ECM components on cell behaviour in vitro. • We compare five different cell types, using the same culture conditions. • The ECM significantly guides all cell responses. • Cell behaviour follows a cell specific ligand-priority ranking. • This gives insight in tissue formation and is essential for biomedical applications.

  6. The Role of Mast Cells in Irritable Bowel Syndrome

    Directory of Open Access Journals (Sweden)

    Kang Nyeong Lee

    2016-01-01

    Full Text Available Irritable bowel syndrome (IBS is one of the most common functional gastrointestinal disorders, but its treatment is unsatisfactory as its pathophysiology is multifactorial. The putative factors of IBS pathophysiology are visceral hypersensitivity and intestinal dysmotility, also including psychological factors, dysregulated gut-brain axis, intestinal microbiota alterations, impaired intestinal permeability, and mucosal immune alterations. Recently, mucosal immune alterations have received much attention with the role of mast cells in IBS. Mast cells are abundant in the intestines and function as intestinal gatekeepers at the interface between the luminal environment in the intestine and the internal milieu under the intestinal epithelium. As a gatekeeper at the interface, mast cells communicate with the adjacent cells such as epithelial, neuronal, and other immune cells throughout the mediators released when they themselves are activated. Many studies have suggested that mast cells play a role in the pathophysiology of IBS. This review will focus on studies of the role of mast cell in IBS and the limitations of studies and will also consider future directions.

  7. Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

    Directory of Open Access Journals (Sweden)

    Silvia Rodríguez-Lombardero

    2014-07-01

    Full Text Available Sky1 is the only member of the SR (Serine–Arginine protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Scope: Structural-based recognition of foreign molecules is essential for activation of dendritic cells (DCs) that play a key role in regulation of gut mucosal immunity. Orally ingested non-starch polysaccharides (NSP) are ascribed many health-promoting properties, but currently we lack insight i.......Conclusions: Collectively, this comparative study revealed that some plant-derived NSP besides those of microbial origin exert modulation of the DC phenotype, with the exact structure being important for the activity.......Scope: Structural-based recognition of foreign molecules is essential for activation of dendritic cells (DCs) that play a key role in regulation of gut mucosal immunity. Orally ingested non-starch polysaccharides (NSP) are ascribed many health-promoting properties, but currently we lack insight...... into the impact of structure and size for their capacity to affect immune responses.Methods and results: This study addresses the importance of chemical structure, size, origin and presence of contaminants for the capacity of both dietary and non-food NSP to modulate DC. Of 28 NSP products, β-glucans of microbial...

  9. Role of Jnk1 in development of neural precursors revealed by iPSC modeling.

    Science.gov (United States)

    Zhang, Qian; Mao, Jian; Zhang, Xiaoxi; Fu, Haifeng; Xia, Siyuan; Yin, Zhinan; Liu, Lin

    2016-09-20

    Jnk1-deficient mice manifest disrupted anterior commissure formation and loss of axonal and dendritic microtubule integrity. However, the mechanisms and the specific stages underlying the developmental defects remain to be elucidated. Here, we report the generation of Jnk1-deficient (Jnk1 KO) iPSCs from Jnk1 KO mouse tail-tip fibroblasts (TTFs) for modeling the neural disease development. The efficiency in the early induction of iPSCs was higher from Jnk1 KO fibroblasts than that of wild-type (WT) fibroblasts. These Jnk1 KO iPSCs exhibited pluripotent stem cell properties and had the ability of differentiation into general three embryonic germ layers in vitro and in vivo. However, Jnk1 KO iPSCs showed reduced capacity in neural differentiation in the spontaneous differentiation by embryoid body (EB) formation. Notably, by directed lineage differentiation, Jnk1 KO iPSCs specifically exhibited an impaired ability to differentiate into early stage neural precursors. Furthermore, the neuroepitheliums generated from Jnk1 KO iPSCs appeared smaller, indicative of neural stem cell developmental defects, as demonstrated by teratoma tests in vivo. These data suggest that Jnk1 deficiency inhibits the development of neural stem cells/precursors and provide insights to further understanding the complex pathogenic mechanisms of JNK1-related neural diseases.

  10. Role of extracellular cations in cell motility, polarity, and chemotaxis

    Directory of Open Access Journals (Sweden)

    Soll D

    2011-04-01

    Full Text Available David R Soll1, Deborah Wessels1, Daniel F Lusche1, Spencer Kuhl1, Amanda Scherer1, Shawna Grimm1,21Monoclonal Antibody Research Institute, Developmental Studies, Hybridoma Bank, Department of Biology, University of Iowa, Iowa City; 2Mercy Medical Center, Surgical Residency Program, Des Moines, Iowa, USAAbstract: The concentration of cations in the aqueous environment of free living organisms and cells within the human body influence motility, shape, and chemotaxis. The role of extracellular cations is usually perceived to be the source for intracellular cations in the process of homeostasis. The role of surface molecules that interact with extracellular cations is believed to be that of channels, transporters, and exchangers. However, the role of Ca2+ as a signal and chemoattractant and the discovery of the Ca2+ receptor have demonstrated that extracellular cations can function as signals at the cell surface, and the plasma membrane molecules they interact with can function as bona fide receptors that activate coupled signal transduction pathways, associated molecules in the plasma membrane, or the cytoskeleton. With this perspective in mind, we have reviewed the cationic composition of aqueous environments of free living cells and cells that move in multicellular organisms, most notably humans, the range of molecules interacting with cations at the cell surface, the concept of a cell surface cation receptor, and the roles extracellular cations and plasma membrane proteins that interact with them play in the regulation of motility, shape, and chemotaxis. Hopefully, the perspective of this review will increase awareness of the roles extracellular cations play and the possibility that many of the plasma membrane proteins that interact with them could also play roles as receptors.Keywords: extracellular cations, chemotaxis, transporters, calcium, receptors

  11. Directing traffic on the NKT-cell highway: a key role for ThPOK.

    Science.gov (United States)

    Chakravarti, Sumone; Godfrey, Dale I

    2010-09-01

    CD1d-restricted NKT cells include CD4(+) and DN subsets, with an additional CD8(+) subset that is present in humans but not in mice. The molecular regulation of CD4/CD8 expression by NKT cells, and differentiation of these NKT-cell subsets, is poorly understood. The transcription factors GATA3 and ThPOK regulate lineage commitment of conventional MHC class II-restricted CD4(+) T cells; however, their role in CD4/CD8 expression by CD1d-restricted NKT cells is less clear. A new study in this issue of the European Journal of Immunology demonstrates a key role for ThPOK in differentiation of NKT-cell subsets. This study reveals that GATA3 and ThPOK are necessary for the development of CD4(+) NKT cells. Furthermore, ThPOK-deficient mice generate an unusual population of CD8(+) NKT cells, which are absent in control mice. This study sheds new light on the underlying molecular events leading to the emergence of distinct NKT-cell subsets.

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

    Science.gov (United States)

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

    2013-04-10

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

  13. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking

    Science.gov (United States)

    Lu, Yi-Ju; Schornack, Sebastian; Spallek, Thomas; Geldner, Niko; Chory, Joanne; Schellmann, Swen; Schumacher, Karin; Kamoun, Sophien; Robatzek, Silke

    2016-01-01

    Summary Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes. PMID:22233428

  14. Genetic characterization of ebi reveals its critical role in Drosophila wing growth

    Science.gov (United States)

    Walker, Cherryl D; Orme, Mariam H; Leevers, Sally J

    2011-01-01

    The ebi gene of Drosophila melanogaster has been implicated in diverse signaling pathways, cellular functions and developmental processes. However, a thorough genetic analysis of this gene has been lacking and the true extent of its biological roles is unclear. Here, we characterize eleven ebi mutations and find that ebi has a novel role in promoting growth of the wing imaginal disc: viable combinations of mutant alleles give rise to adults with small wings. Wing discs with reduced EBI levels are correspondingly small and exhibit downregulation of Notch target genes. Furthermore, we show that EBI colocalizes on polytene chromosomes with Smrter (SMR), a transcriptional corepressor, and Suppressor of Hairless (SU(H)), the primary transcription factor involved in Notch signaling. Interestingly, the mammalian orthologs of ebi, transducin β-like 1 (TBL1) and TBL-related 1 (TBLR1), function as corepressor/coactivator exchange factors and are required for transcriptional activation of Notch target genes. We hypothesize that EBI acts to activate (de-repress) transcription of Notch target genes important for Drosophila wing growth by functioning as a corepressor/coactivator exchange factor for SU(H). PMID:22041576

  15. Solute restriction reveals an essential role for clag3-associated channels in malaria parasite nutrient acquisition.

    Science.gov (United States)

    Pillai, Ajay D; Nguitragool, Wang; Lyko, Brian; Dolinta, Keithlee; Butler, Michelle M; Nguyen, Son T; Peet, Norton P; Bowlin, Terry L; Desai, Sanjay A

    2012-12-01

    The plasmodial surface anion channel (PSAC) increases erythrocyte permeability to many solutes in malaria but has uncertain physiological significance. We used a PSAC inhibitor with different efficacies against channels from two Plasmodium falciparum parasite lines and found concordant effects on transport and in vitro parasite growth when external nutrient concentrations were reduced. Linkage analysis using this growth inhibition phenotype in the Dd2 × HB3 genetic cross mapped the clag3 genomic locus, consistent with a role for two clag3 genes in PSAC-mediated transport. Altered inhibitor efficacy, achieved through allelic exchange or expression switching between the clag3 genes, indicated that the inhibitor kills parasites through direct action on PSAC. In a parasite unable to undergo expression switching, the inhibitor selected for ectopic homologous recombination between the clag3 genes to increase the diversity of available channel isoforms. Broad-spectrum inhibitors, which presumably interact with conserved sites on the channel, also exhibited improved efficacy with nutrient restriction. These findings indicate that PSAC functions in nutrient acquisition for intracellular parasites. Although key questions regarding the channel and its biological role remain, antimalarial drug development targeting PSAC should be pursued.

  16. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

    Directory of Open Access Journals (Sweden)

    Valeriy Demchev

    Full Text Available Fibrinogen like protein 1(Fgl1 is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.

  17. Automated multidimensional image analysis reveals a role for Abl in embryonic wound repair.

    Science.gov (United States)

    Zulueta-Coarasa, Teresa; Tamada, Masako; Lee, Eun J; Fernandez-Gonzalez, Rodrigo

    2014-07-01

    The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. We introduce MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. We validate MEDUSA by quantifying wound closure in Drosophila embryos, and we show that the results of our automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. We demonstrate that MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, we find that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. We demonstrate that Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein β-catenin at the wound margin during embryonic wound repair. Finally, we discuss different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin.

  18. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    Science.gov (United States)

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  19. Genetic triple dissociation reveals multiple roles for dopamine in reinforcement learning.

    Science.gov (United States)

    Frank, Michael J; Moustafa, Ahmed A; Haughey, Heather M; Curran, Tim; Hutchison, Kent E

    2007-10-09

    What are the genetic and neural components that support adaptive learning from positive and negative outcomes? Here, we show with genetic analyses that three independent dopaminergic mechanisms contribute to reward and avoidance learning in humans. A polymorphism in the DARPP-32 gene, associated with striatal dopamine function, predicted relatively better probabilistic reward learning. Conversely, the C957T polymorphism of the DRD2 gene, associated with striatal D2 receptor function, predicted the degree to which participants learned to avoid choices that had been probabilistically associated with negative outcomes. The Val/Met polymorphism of the COMT gene, associated with prefrontal cortical dopamine function, predicted participants' ability to rapidly adapt behavior on a trial-to-trial basis. These findings support a neurocomputational dissociation between striatal and prefrontal dopaminergic mechanisms in reinforcement learning. Computational maximum likelihood analyses reveal independent gene effects on three reinforcement learning parameters that can explain the observed dissociations.

  20. Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

    Science.gov (United States)

    Wilson, J. W.; Ott, C. M.; zuBentrup, K. Honer; Ramamurthy R.; Quick, L.; Porwollik, S.; Cheng, P.; McClellan, M.; Tsaprailis, G.; Radabaugh, T.; Hunt, A.; Fernandez, D.; Richter, E.; Shah, M.; Kilcoyne, M.; Joshi, L.; Nelman-Gonzalez, M.; Hing, S.; Parra, M.; Dumaras, P.; Norwood, K.; Nickerson, C. A.; Bober, R.; Devich, J.; Ruggles, A.

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

  1. Genome-wide transcriptional profiling reveals microRNA-correlated genes and biological processes in human lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    Liang Wang

    Full Text Available BACKGROUND: Expression level of many genes shows abundant natural variation in human populations. The variations in gene expression are believed to contribute to phenotypic differences. Emerging evidence has shown that microRNAs (miRNAs are one of the key regulators of gene expression. However, past studies have focused on the miRNA target genes and used loss- or gain-of-function approach that may not reflect natural association between miRNA and mRNAs. METHODOLOGY/PRINCIPAL FINDINGS: To examine miRNA regulatory effect on global gene expression under endogenous condition, we performed pair-wise correlation coefficient analysis on expression levels of 366 miRNAs and 14,174 messenger RNAs (mRNAs in 90 immortalized lymphoblastoid cell lines, and observed significant correlations between the two species of RNA transcripts. We identified a total of 7,207 significantly correlated miRNA-mRNA pairs (false discovery rate q<0.01. Of those, 4,085 pairs showed positive correlations while 3,122 pairs showed negative correlations. Gene ontology analyses on the miRNA-correlated genes revealed significant enrichments in several biological processes related to cell cycle, cell communication and signal transduction. Individually, each of three miRNAs (miR-331, -98 and -33b demonstrated significant correlation with the genes in cell cycle-related biological processes, which is consistent with important role of miRNAs in cell cycle regulation. CONCLUSIONS/SIGNIFICANCE: This study demonstrates feasibility of using naturally expressed transcript profiles to identify endogenous correlation between miRNA and miRNA. By applying this genome-wide approach, we have identified thousands of miRNA-correlated genes and revealed potential role of miRNAs in several important cellular functions. The study results along with accompanying data sets will provide a wealth of high-throughput data to further evaluate the miRNA-regulated genes and eventually in phenotypic variations of

  2. The Role of Cell Compartmentalization and Cell Differentiation in Cyanobacterial Excavation of Miineral Carbonates

    Science.gov (United States)

    Garcia-Pichel, F.; Guida, B. S.; Couradeau, E.

    2015-12-01

    The bioerosion of coastal limestones and biogenic carbonates by boring filamentous or pseudo-filamentous cyanobacteria is not only a geomicrobial phenomenon of global proportions, but also plays an important role in the demise of coral reefs, and affects significantly human enterprises like bivalve fisheries. In spite of its importance, the mechanism by which cyanobacteria excavate carbonates constitutes an apparent paradox, in that their metabolism will tend to precipitate carbonates, not dissolved them. We have previously advanced, and obtained evidence for, a mechanism of excavation that relies on the uptake of Ca2+ by cells at the boring front, its trans-cellular transport along the filaments, and its eventual active excretion at the solid/liquid interface. It was postulated that the mechanism involved the strategically organized deployment of Ca2+ transport enzymes like P-type Ca2+ ATPases and Ca2+ channels. Here we present evidence that confirms this basic mechanism, but also reveals that it is based on an unexpected level of cellular complexity. The model organism Mastigocoleus testarum BC008, transports Ca2+ from the mineral to the external medium using a repetitive, polar arrangement of Ca2+ ATPases, localized preferentially on one cellular pole, in a ring conformation on the cell membrane adjacent to the trans-cellular septum, pumping Ca2+ locally towards the periplasmic space, from which it passively enters the next cell. This strain also develops specialized groups of cells, which we named calcicytes, often but not exclusively located at the ends of filaments, that accumulate large concentrations of Ca2+, some 40-fold higher than typical in microbes, and seem to act as sinks or capacitors in the trans-cellular Ca2+ transport. Calcicytes are also characterized by a lack of photosynthetic pigments, and a very high intracellular pH. These cellular adaptations can also be found in evolutionary distant euendoliths such as the pseudofilamentous Hyella sp.

  3. The Role of Glial Cells in Drug Abuse

    OpenAIRE

    Miguel-Hidalgo, Jose Javier

    2009-01-01

    Neuronal dysfunction in the prefrontal cortex, limbic structures, nucleus accumbens and ventral tegmental area is considered to underlie the general physiopathological mechanisms for substance use disorders. Glutamatergic, dopaminergic and opioidoergic neuronal mechanisms in those brain areas have been targeted in the development of pharmacotherapies for drug abuse and dependence. However, despite the pivotal role of neurons in the mechanisms of addiction, these cells are not the only cell ty...

  4. The role of the vascular dendritic cell network in atherosclerosis

    OpenAIRE

    Alberts-Grill, Noah; Denning, Timothy L.; Rezvan, Amir; Jo, Hanjoong

    2013-01-01

    A complex role has been described for dendritic cells (DCs) in the potentiation and control of vascular inflammation and atherosclerosis. Resident vascular DCs are found in the intima of atherosclerosis-prone vascular regions exposed to disturbed blood flow patterns. Several phenotypically and functionally distinct vascular DC subsets have been described. The functional heterogeneity of these cells and their contributions to vascular homeostasis, inflammation, and atherosclerosis are only rec...

  5. Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

    Science.gov (United States)

    Barbazuk, W. Brad

    2017-01-01

    RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates. PMID:28242684

  6. Human exceptional longevity: transcriptome from centenarians is distinct from septuagenarians and reveals a role of Bcl-xL in successful aging

    Science.gov (United States)

    Borras, Consuelo; M. Abdelaziz, Kheira; Gambini, Juan; Serna, Eva; Inglés, Marta; de la Fuente, Monica; Garcia, Idoia; Matheu, Ander; Sanchís, Paula; Belenguer, Angel; Errigo, Alessandra; Avellana, Juan- Antonio; Barettino, Ana; Lloret-Fernández, Carla; Flames, Nuria; Pes, Gianni; Rodriguez-Mañas, Leocadio; Viña, Jose

    2016-01-01

    Centenarians not only enjoy an extraordinary aging, but also show a compression of morbidity. Using functional transcriptomic analysis of peripheral blood mononuclear cells (PMBC) we identified 1721 mRNAs differentially expressed by centenarians when compared with septuagenarians and young people. Sub-network analysis led us to identify Bcl - xL as an important gene up-regulated in centenarians. It is involved in the control of apoptosis, cellular damage protection and also in modulation of immune response, all associated to healthy aging. Indeed, centenarians display lower plasma cytochrome C levels, higher mitochondrial membrane potential and also less cellular damage accumulation than septuagenarians. Leukocyte chemotaxis and NK cell activity are significantly impaired in septuagenarians compared with young people whereas centenarians maintain them. To further ascertain the functional role of Bcl- xL in cellular aging, we found that lymphocytes from septuagenarians transduced with Bcl-xL display a reduction in senescent-related markers. Finally, to demonstrate the role of BcL-xL in longevity at the organism level, C. elegans bearing a gain of function mutation in the BcL-xL ortholog ced-9, showed a significant increase in mean and maximal life span. These results show that mRNA expression in centenarians is unique and reveals that BcL- xL plays an important role in exceptional aging. PMID:27794564

  7. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    DEFF Research Database (Denmark)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

    2012-01-01

    Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells gener...... cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.......Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells...... generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine...

  8. Gene expression analysis in citrus reveals the role of gibberellins on photosynthesis and stress.

    Science.gov (United States)

    Huerta, Laura; Forment, Javier; Gadea, José; Fagoaga, Carmen; Peña, Leandro; Pérez-Amador, Miguel A; García-Martínez, José Luis

    2008-11-01

    The effect of gibberellins (GA) on internode transcriptome was investigated in transgenic Carrizo citrange (Citrus sinensis x Poncirus trifoliata) plants overexpressing endogenous CcGA20ox1 (encoding a GA biosynthetic gene), and in non-transformed explants treated with GA(3), using a citrus cDNA microarray. Substantial modulation of gene expression was found in sense CcGA20ox plants. Extensive up-regulation of genes involved in photosynthesis and carbon utilization, and down-regulation of those involved in protein synthesis and ribosome biogenesis were shown for the first time in plants with higher GA content. Importantly, increase of net photosynthesis in attached leaves was also demonstrated. Expression of other genes belonging to functional groups not reported previously to be regulated by GA (mainly abiotic and biotic stresses, and cuticle biosynthesis), and genes involved in cell division and cell wall architecture were also differentially expressed. Culture of citrus explants for 24 h in GA(3) solution produced much lower changes in the transcriptome compared with CcGA20ox plants (1.6% versus 16%, respectively, of total genes in the microarray), suggesting that most of the changes observed in CcGA20ox plants were a consequence of a long-standing GA effect. Interestingly, genes related to abiotic and biotic stresses were similarly modulated in transgenics and GA(3)-treated explants.

  9. The role of ammonites in the Mesozoic marine food web revealed by jaw preservation.

    Science.gov (United States)

    Kruta, Isabelle; Landman, Neil; Rouget, Isabelle; Cecca, Fabrizio; Tafforeau, Paul

    2011-01-07

    Ammonites are prominent in macroevolutionary studies because of their abundance and diversity in the fossil record, but their paleobiology and position in the marine food web are not well understood due to the lack of preserved soft tissue. We present three-dimensional reconstructions of the buccal apparatus in the Mesozoic ammonite Baculites with the use of synchrotron x-ray microtomography. Buccal mass morphology, combined with the coexistence of food remains found in the buccal mass, suggests that these ammonites fed on plankton. This diet may have extended to all aptychophoran ammonites, which share the same buccal mass morphology. Understanding the role of these ammonites in the Mesozoic food web provides insights into their radiation in the Early Jurassic, as well as their extinction at the end of the Cretaceous/early Paleogene.

  10. Quantitative Lipoproteomics in Clostridium difficile Reveals a Role for Lipoproteins in Sporulation.

    Science.gov (United States)

    Charlton, Thomas M; Kovacs-Simon, Andrea; Michell, Stephen L; Fairweather, Neil F; Tate, Edward W

    2015-11-19

    Bacterial lipoproteins are surface exposed, anchored to the membrane by S-diacylglyceryl modification of the N-terminal cysteine thiol. They play important roles in many essential cellular processes and in bacterial pathogenesis. For example, Clostridium difficile is a Gram-positive anaerobe that causes severe gastrointestinal disease; however, its lipoproteome remains poorly characterized. Here we describe the application of metabolic tagging with alkyne-tagged lipid analogs, in combination with quantitative proteomics, to profile protein lipidation across diverse C. difficile strains and on inactivation of specific components of the lipoprotein biogenesis pathway. These studies provide the first comprehensive map of the C. difficile lipoproteome, demonstrate the existence of two active lipoprotein signal peptidases, and provide insights into lipoprotein function, implicating the lipoproteome in transmission of this pathogen.

  11. Revealing Fundamental Interactions: the Role of Polarized Positrons and Electrons at the Linear Collider

    Energy Technology Data Exchange (ETDEWEB)

    Moortgat-Pick, G.; /CERN /Durham U., IPPP; Abe, T.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke,; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, T.; Eberl, H.; Ellis, John R.; Flottman, K.; Frass, H.; /CERN /Durham U., IPPP /Colorado U. /Tel-Aviv

    2005-07-06

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.

  12. The Role of polarized positrons and electrons in revealing fundamental interactions at the linear collider

    Energy Technology Data Exchange (ETDEWEB)

    Moortgat-Pick, G.; /CERN /Durham U., IPPP; Abe, T.; Alexander, G.; Ananthanarayan, B.; Babich, A.A.; Bharadwaj, V.; Barber, D.; Bartl, A.; Brachmann, A.; Chen, S.; Clarke,; Clendenin, J.E.; Dainton, J.; Desch, K.; Diehl, M.; Dobos, B.; Dorland, T.; Eberl, H.; Ellis, John R.; Flottman, K.; Frass, H.; /CERN /Durham U., IPPP /Colorado U. /Tel-Aviv

    2005-07-01

    The proposed International Linear Collider (ILC) is well-suited for discovering physics beyond the Standard Model and for precisely unraveling the structure of the underlying physics. The physics return can be maximized by the use of polarized beams. This report shows the paramount role of polarized beams and summarizes the benefits obtained from polarizing the positron beam, as well as the electron beam. The physics case for this option is illustrated explicitly by analyzing reference reactions in different physics scenarios. The results show that positron polarization, combined with the clean experimental environment provided by the linear collider, allows to improve strongly the potential of searches for new particles and the identification of their dynamics, which opens the road to resolve shortcomings of the Standard Model. The report also presents an overview of possible designs for polarizing both beams at the ILC, as well as for measuring their polarization.

  13. Role of stem/progenitor cells in reparative disorders

    Directory of Open Access Journals (Sweden)

    Pretheeban Thavaneetharajah

    2012-12-01

    Full Text Available Abstract Adult stem cells are activated to proliferate and differentiate during normal tissue homeostasis as well as in disease states and injury. This activation is a vital component in the restoration of function to damaged tissue via either complete or partial regeneration. When regeneration does not fully occur, reparative processes involving an overproduction of stromal components ensure the continuity of tissue at the expense of its normal structure and function, resulting in a “reparative disorder”. Adult stem cells from multiple organs have been identified as being involved in this process and their role in tissue repair is being investigated. Evidence for the participation of mesenchymal stromal cells (MSCs in the tissue repair process across multiple tissues is overwhelming and their role in reparative disorders is clearly demonstrated, as is the involvement of a number of specific signaling pathways. Transforming growth factor beta, bone morphogenic protein and Wnt pathways interact to form a complex signaling network that is critical in regulating the fate choices of both stromal and tissue-specific resident stem cells (TSCs, determining whether functional regeneration or the formation of scar tissue follows an injury. A growing understanding of both TSCs, MSCs and the complex cascade of signals regulating both cell populations have, therefore, emerged as potential therapeutic targets to treat reparative disorders. This review focuses on recent advances on the role of these cells in skeletal muscle, heart and lung tissues.

  14. Non-neuronal Cells in ALS: Role of Glial, Immune cells and Blood-CNS Barriers.

    Science.gov (United States)

    Puentes, Fabiola; Malaspina, Andrea; van Noort, Johannes M; Amor, Sandra

    2016-03-01

    Neurological dysfunction and motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is strongly associated with neuroinflammation reflected by activated microglia and astrocytes in the CNS. In ALS endogenous triggers in the CNS such as aggregated protein and misfolded proteins activate a pathogenic response by innate immune cells. However, there is also strong evidence for a neuroprotective immune response in ALS. Emerging evidence also reveals changes in the peripheral adaptive immune responses as well as alterations in the blood brain barrier that may aid traffic of lymphocytes and antibodies into the CNS. Understanding the triggers of neuroinflammation is key to controlling neuronal loss. Here, we review the current knowledge regarding the roles of non-neuronal cells as well as the innate and adaptive immune responses in ALS. Existing ALS animal models, in particular genetic rodent models, are very useful to study the underlying pathogenic mechanisms of motor neuron degeneration. We also discuss the approaches used to target the pathogenic immune responses and boost the neuroprotective immune pathways as novel immunotherapies for ALS.

  15. Role of hepatocyte nuclear factor 4α (HNF4α) in cell proliferation and cancer.

    Science.gov (United States)

    Walesky, Chad; Apte, Udayan

    2015-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.

  16. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Directory of Open Access Journals (Sweden)

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  17. An Oncogenic Role for Alternative NF-κB Signaling in DLBCL Revealed upon Deregulated BCL6 Expression

    Directory of Open Access Journals (Sweden)

    Baochun Zhang

    2015-05-01

    Full Text Available Diffuse large B cell lymphoma (DLBCL is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-κB activity, a role for oncogenic lesions that activate the alternative NF-κB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-κB pathway, occurs in ∼15% of DLBCLs and that it often coexists with BCL6 translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-κB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-κB pathway in DLBCL development.

  18. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    Science.gov (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  19. The Structure of Neurexin 1[alpha] Reveals Features Promoting a Role as Synaptic Organizer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fang; Venugopal, Vandavasi; Murray, Beverly; Rudenko, Gabby (Michigan)

    2014-10-02

    {alpha}-Neurexins are essential synaptic adhesion molecules implicated in autism spectrum disorder and schizophrenia. The {alpha}-neurexin extracellular domain consists of six LNS domains interspersed by three EGF-like repeats and interacts with many different proteins in the synaptic cleft. To understand how {alpha}-neurexins might function as synaptic organizers, we solved the structure of the neurexin 1{alpha} extracellular domain (n1{alpha}) to 2.65 {angstrom}. The L-shaped molecule can be divided into a flexible repeat I (LNS1-EGF-A-LNS2), a rigid horseshoe-shaped repeat II (LNS3-EGF-B-LNS4) with structural similarity to so-called reelin repeats, and an extended repeat III (LNS5-EGF-B-LNS6) with controlled flexibility. A 2.95 {angstrom} structure of n1{alpha} carrying splice insert SS3 in LNS4 reveals that SS3 protrudes as a loop and does not alter the rigid arrangement of repeat II. The global architecture imposed by conserved structural features enables {alpha}-neurexins to recruit and organize proteins in distinct and variable ways, influenced by splicing, thereby promoting synaptic function.

  20. Comparative expression profiling reveals an essential role for raldh2 in epimorphic regeneration.

    Science.gov (United States)

    Mathew, Lijoy K; Sengupta, Sumitra; Franzosa, Jill A; Perry, Jessica; La Du, Jane; Andreasen, Eric A; Tanguay, Robert L

    2009-11-27

    Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.

  1. Simulations reveal the role of composition into the atomic-level flexibility of bioactive glass cements.

    Science.gov (United States)

    Tian, Kun Viviana; Chass, Gregory A; Di Tommaso, Devis

    2016-01-14

    Bioactive glass ionomer cements (GICs), the reaction product of a fluoro-alumino-silicate glass and polyacrylic acid, have been in effective use in dentistry for over 40 years and more recently in orthopaedics and medical implantation. Their desirable properties have affirmed GIC's place in the medical materials community, yet are limited to non-load bearing applications due to the brittle nature of the hardened composite cement, thought to arise from the glass component and the interfaces it forms. Towards helping resolve the fundamental bases of the mechanical shortcomings of GICs, we report the 1st ever computational models of a GIC-relevant component. Ab initio molecular dynamics simulations were employed to generate and characterise three fluoro-alumino-silicate glasses of differing compositions with focus on resolving the atomic scale structural and dynamic contributions of aluminium, phosphorous and fluorine. Analyses of the glasses revealed rising F-content leading to the expansion of the glass network, compression of Al-F bonding, angular constraint at Al-pivots, localisation of alumino-phosphates and increased fluorine diffusion. Together, these changes to the structure, speciation and dynamics with raised fluorine content impart an overall rigidifying effect on the glass network, and suggest a predisposition to atomic-level inflexibility, which could manifest in the ionomer cements they form.

  2. Structure of Est3 reveals a bimodal surface with differential roles in telomere replication.

    Science.gov (United States)

    Rao, Timsi; Lubin, Johnathan W; Armstrong, Geoffrey S; Tucey, Timothy M; Lundblad, Victoria; Wuttke, Deborah S

    2014-01-01

    Telomerase is essential for continuous cellular proliferation. Substantial insights have come from studies of budding yeast telomerase, which consists of a catalytic core in association with two regulatory proteins, ever shorter telomeres 1 and 3 (Est1 and Est3). We report here a high-resolution structure of the Est3 telomerase subunit determined using a recently developed strategy that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithms. Est3 adopts an overall protein fold which is structurally similar to that adopted by the shelterin component TPP1. However, the characteristics of the surface of the experimentally determined Est3 structure are substantially different from those predicted by prior homology-based models of Est3. Structure-guided mutagenesis of the complete surface of the Est3 protein reveals two adjacent patches on a noncanonical face of the protein that differentially mediate telomere function. Mapping these two patches on the Est3 structure defines a set of shared features between Est3 and HsTPP1, suggesting an analogous multifunctional surface on TPP1.

  3. mRNA expression profiling reveals a role of Helicobacter pylorivacuolating toxin in escaping host defense

    Institute of Scientific and Technical Information of China (English)

    Jian-Ping Yuan; Tao Li; Zhen-Hong Li; Gui-Zhen Yang; Bao-Yu Hu; Xiao-Dong Shi; Tie-Liu Shi; Shan-Qing Tong; Xiao-Kui Guo

    2004-01-01

    AIM: To study the immune response of host to Helicobacter pylori VacA.METHODS: The monocyte/macrophage-like U937 cells were infected with Helicobacter pylori vacA-positive strain NCTC 11638 or isogenic vacA-negative mutant. Differentially expressed genes were identified at 2, 6, 10, and 24 h postinfection by cDNA microarray. Differential expressions of some genes were confirmed by Northern blot.RESULTS: More than 100 genes altered their mRNA expression at different time points respectively, many of which were identified to be related to immune evasion.CONCLUSION: VacA is a crucial element for H pylorito escape from host immune defense by means of differentially regulating the expression of some related genes. These genes, previously known or unknown to be involved in the mechanism of immune evasion, deserve further investigation to unearth much more information complicated in the immune response.

  4. A male with unilateral microphthalmia reveals a role for TMX3 in eye development

    DEFF Research Database (Denmark)

    Chao, Ryan; Nevin, Linda; Agarwal, Pooja;

    2010-01-01

    of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male...... with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye...... development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained...

  5. Conserved BK channel-protein interactions reveal signals relevant to cell death and survival.

    Directory of Open Access Journals (Sweden)

    Bernd Sokolowski

    Full Text Available The large-conductance Ca(2+-activated K(+ (BK channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP, stathmin (STMN, cortactin (CTTN, and prohibitin (PHB, of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca(2+ binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt, glycogen synthase kinase-3β (GSK3β and phosphoinositide-dependent kinase-1 (PDK1. Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite

  6. Formalin-induced fluorescence reveals cell shape and morphology in biological tissue samples.

    Directory of Open Access Journals (Sweden)

    Ulrich Leischner

    Full Text Available Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques.

  7. RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival[W

    Science.gov (United States)

    González-Ballester, David; Casero, David; Cokus, Shawn; Pellegrini, Matteo; Merchant, Sabeeha S.; Grossman, Arthur R.

    2010-01-01

    The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival. PMID:20587772

  8. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

    Science.gov (United States)

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A; Irudayaraj, Joseph M K

    2015-02-01

    Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

  9. Critical roles of mTOR Complex 1 and 2 for T follicular helper cell differentiation and germinal center responses.

    Science.gov (United States)

    Yang, Jialong; Lin, Xingguang; Pan, Yun; Wang, Jinli; Chen, Pengcheng; Huang, Hongxiang; Xue, Hai-Hui; Gao, Jimin; Zhong, Xiao-Ping

    2016-09-30

    T follicular helper (Tfh) cells play critical roles for germinal center responses and effective humoral immunity. We report here that mTOR in CD4 T cells is essential for Tfh differentiation. In Mtor(f/f)-Cd4Cre mice, both constitutive and inducible Tfh differentiation is severely impaired, leading to defective germinal center B cell formation and antibody production. Moreover, both mTORC1 and mTORC2 contribute to Tfh and GC B cell development but may do so via distinct mechanisms. mTORC1 mainly promotes CD4 T cell proliferation to reach the cell divisions necessary for Tfh differentiation, while Rictor/mTORC2 regulates Tfh differentiation by promoting Akt activation and TCF1 expression without grossly influencing T cell proliferation. Together, our results reveal crucial but distinct roles for mTORC1 and mTORC2 in CD4 T cells during Tfh differentiation and germinal center responses.

  10. [The role of IRA B cells in selected inflammatory processes].

    Science.gov (United States)

    Zasada, Magdalena; Rutkowska-Zapała, Magdalena; Lenart, Marzena; Kwinta, Przemko

    2016-03-16

    The first report about the discovery of new, previously unknown immune cells named IRA B cells (innate response activator B cells) appeared in 2012. So far, their presence has been verified in both mice and humans. However, IRA B cells belong to the family of B lymphocytes and have a number of characteristics unique to this group of cells. IRA B cells are formed from activated B1a lymphocytes after their contact with a pathogen. B1a lymphocytes mainly reside within body cavities. Activated by the pathogen, they move on into secondary lymphoid organs (spleen, lymph nodes) where they differentiate into IRA B cells. IRA B cells are a rich source of granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF can stimulate IRA B cells in an autocrine manner for the secretion of intracellular stocks of immunoglobulin M (IgM), which can facilitate pathogens' phagocytosis by neutrophils. GM-CSF also stimulates neutrophils into active phagocytosis. Rapid eradication of the pathogen can prevent the development of an excessive inflammatory response, which can be dangerous for the organism. Until now the involvement of IRA B lymphocytes in the pathogenesis of sepsis and pneumonia has been proven, as well as their role in the progression of atherosclerotic lesions in mice. There is research in progress on the possibility of increasing the number of IRA B cells, for example by intravenous supply of modified immunoglobulins. It is necessary to characterize human IRA B cells and to determine their role in the functioning of the immune system.

  11. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G

    2011-01-01

    Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC...... (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...... rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view...

  12. Expression and Fuactional Role of HERG1, K+ Channels in Leukemic Cells and Leukemic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    LI Huiyu; LIU Liqiong; GUO Tiannan; ZHANG Jiahua; LI Xiaoqing; DU Wen; LIU Wei; CHEN Xiangjun; HUANG Shi'ang

    2007-01-01

    In order to investigate the expression and functional role of HERG1 K+ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K+ channels expression in leukemic cells and LSCs. The functional role of HERG1 K+ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34+/CD38-, CD123+ LSCs but not in circulating CD34+ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K+ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K+ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K+ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy.

  13. The role of satellite cells in muscle hypertrophy.

    Science.gov (United States)

    Blaauw, Bert; Reggiani, Carlo

    2014-02-01

    The role of satellite cells in muscle hypertrophy has long been a debated issue. In the late 1980s it was shown that proteins remain close to the myonucleus responsible for its synthesis, giving rise to the idea of a nuclear domain. This, together with the observation that during various models of muscle hypertrophy there is an activation of the muscle stem cells, i.e. satellite cells, lead to the idea that satellite cell activation is required for muscle hypertrophy. Thus, satellite cells are not only responsible for muscle repair and regeneration, but also for hypertrophic growth. Further support for this line of thinking was obtained after studies showing that irradiation of skeletal muscle, and therefore elimination of all satellite cells, completely prevented overload-induced hypertrophy. Recently however, using different transgenic approaches, it has become clear that muscle hypertrophy can occur without a contribution of satellite cells, even though in most situations of muscle hypertrophy satellite cells are activated. In this review we will discuss the contribution of satellite cells, and other muscle-resident stem cells, to muscle hypertrophy both in mice as well as in humans.

  14. Inflammatory role of the acinar cells during acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Isabel; De; Dios

    2010-01-01

    Pancreatic acinar cells are secretory cells whose main function is to synthesize, store and f inally release digestive enzymes into the duodenum. However, in response to noxious stimuli, acinar cells behave like real inflammatory cells because of their ability to activate signalling transduction pathways involved in the expression of inflammatory mediators. Mediated by the kinase cascade, activation of Nuclear factor-κB, Activating factor-1 and Signal transducers and activators of transcription transcription factors has been demonstrated in acinar cells, resulting in overexpression of inflammatory genes. In turn, kinase activity is down-regulated by protein phosphatases and the f inal balance between kinase and phosphatase activity will determine the capability of the acinar cells to produce inflammatory factors. The kinase/ phosphatase pair is a redox-sensitive system in which kinase activation overwhelms phosphatase activity under oxidant conditions. Thus, the oxidative stress developed within acinar cells at early stages of acute pancreatitis triggers the activation of signalling pathways involved in the up-regulation of cytokines, chemokines and adhesion molecules. In this way, acinar cells trigger the release of the f irst inflammatory signals which can mediate the activation and recruitment of circulating inflammatorycells into the injured pancreas. Accordingly, the role of acinar cells as promoters of the inflammatory response in acute pancreatitis may be considered. This concept leads to amplifying the focus from leukocyte to acinar cells themselves, to explain the local inflammation in early pancreatitis.

  15. [The role of regulatory T cells in allergic contact dermatitis].

    Science.gov (United States)

    Krecisz, Beata; Chomiczewska, Dorota; Kieć-Swierczyńska, Marta

    2009-01-01

    Regulatory T cells (Treg) play a crucial role in the regulatory mechanisms of immune system. They are responsible for the induction and maintenance of immune tolerance. They are also involved in the pathogenesis of autoimmune and allergic diseases and implicated in transplant rejection and immunopathology of cancers. Treg cells constitute the population of lymphocytes heterogeneous in their phenotype. CD4+CD25+Foxp3+ cells are most important among Treg subsets. Modification of Treg actvity may be useful in the therapy of different diseases, including allergic disorders.

  16. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    Science.gov (United States)

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  17. Genetic Disruption of 2-Arachidonoylglycerol Synthesis Reveals a Key Role for Endocannabinoid Signaling in Anxiety Modulation

    Directory of Open Access Journals (Sweden)

    Brian C. Shonesy

    2014-12-01

    Full Text Available Endocannabinoid (eCB signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders.

  18. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Directory of Open Access Journals (Sweden)

    Cristian Del Campo

    2015-10-01

    Full Text Available Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  19. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Science.gov (United States)

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  20. Disease-aging network reveals significant roles of aging genes in connecting genetic diseases.

    Science.gov (United States)

    Wang, Jiguang; Zhang, Shihua; Wang, Yong; Chen, Luonan; Zhang, Xiang-Sun

    2009-09-01

    One of the challenging problems in biology and medicine is exploring the underlying mechanisms of genetic diseases. Recent studies suggest that the relationship between genetic diseases and the aging process is important in understanding the molecular mechanisms of complex diseases. Although some intricate associations have been investigated for a long time, the studies are still in their early stages. In this paper, we construct a human disease-aging network to study the relationship among aging genes and genetic disease genes. Specifically, we integrate human protein-protein interactions (PPIs), disease-gene associations, aging-gene associations, and physiological system-based genetic disease classification information in a single graph-theoretic framework and find that (1) human disease genes are much closer to aging genes than expected by chance; and (2) diseases can be categorized into two types according to their relationships with aging. Type I diseases have their genes significantly close to aging genes, while type II diseases do not. Furthermore, we examine the topological characters of the disease-aging network from a systems perspective. Theoretical results reveal that the genes of type I diseases are in a central position of a PPI network while type II are not; (3) more importantly, we define an asymmetric closeness based on the PPI network to describe relationships between diseases, and find that aging genes make a significant contribution to associations among diseases, especially among type I diseases. In conclusion, the network-based study provides not only evidence for the intricate relationship between the aging process and genetic diseases, but also biological implications for prying into the nature of human diseases.

  1. Global phylogeography of Dengue type 1 and 2 viruses reveals the role of India.

    Science.gov (United States)

    Walimbe, Atul M; Lotankar, Mrunalini; Cecilia, D; Cherian, Sarah S

    2014-03-01

    Patterns in virus dispersal and epidemiology of viral diseases can be revealed by phylogeographic studies. Currently knowledge about phylogeography of Dengue virus (DENV) Types 1 and 2 is limited. We carried out the phylogeographic analyses for DENV-1 and DENV-2, by the Bayesian Markov Chain Monte Carlo (MCMC) approach, with emphasis on Indian isolates in relation to the global evolutionary dynamics of the viruses. More than 250 E-gene sequences of each virus, available in GenBank, were used for the analyses. The study was focused on understanding the most likely geographical origin for the major genotypes and sub-lineages of DENV-1/DENV-2 and also the possible pathways in the dispersal of the virus. The results showed that for DENV-1, Southeast Asia was the most likely geographical origin and India was determined to be the ancestral location of the Cosmopolitan genotype circulating in India, Sri Lanka, West and East Africa, Caribbean region, East and Southeast Asia. For DENV-2, the ancestral source could not be precisely inferred. Further, in spite of the earliest isolate from Trinidad-1953 of the American genotype, it was depicted that India may have been the probable ancestor of this genotype. India was also determined to be the ancestral location of a subgroup of the Cosmopolitan genotype. It was noted that DENV-1 and DENV-2 were introduced into India during 1940s and 1910s respectively. Subsequently, dispersal of both the viruses between India and different regions including West, East and Central Africa, Southeast and East Asia and Caribbean was inferred. Overall, the current study provides insight into the spatial as well as temporal dynamics of dengue virus serotypes 1 and 2.

  2. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

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

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

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

  4. EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

    Science.gov (United States)

    Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

    2016-01-01

    Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

  5. Critical roles for lipomannan and lipoarabinomannan in cell wall integrity of mycobacteria and pathogenesis of tuberculosis.

    Science.gov (United States)

    Fukuda, Takeshi; Matsumura, Takayuki; Ato, Manabu; Hamasaki, Maho; Nishiuchi, Yukiko; Murakami, Yoshiko; Maeda, Yusuke; Yoshimori, Tamotsu; Matsumoto, Sohkichi; Kobayashi, Kazuo; Kinoshita, Taroh; Morita, Yasu S

    2013-02-19

    Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a

  6. Role of neural precursor cells in promoting repair following stroke

    Institute of Scientific and Technical Information of China (English)

    Pooya DIBAJNIA; Cindi M MORSHEAD

    2013-01-01

    Stem cell-based therapies for the treatment of stroke have received considerable attention.Two broad approaches to stem cell-based therapies have been taken:the transplantation of exogenous stem cells,and the activation of endogenous neural stem and progenitor cells (together termed neural precursors).Studies examining the transplantation of exogenous cells have demonstrated that neural stem and progenitor cells lead to the most clinically promising results.Endogenous activation of neural precursors has also been explored based on the fact that resident precursor cells have the inherent capacity to proliferate,migrate and differentiate into mature neurons in the uninjured adult brain.Studies have revealed that these neural precursor cell behaviours can be activated following stroke,whereby neural precursors will expand in number,migrate to the infarct site and differentiate into neurons.However,this innate response is insufficient to lead to functional recovery,making it necessary to enhance the activation of endogenous precursors to promote tissue repair and functional recovery.Herein we will discuss the current state of the stem cell-based approaches with a focus on endogenous repair to treat the stroke injured brain.

  7. Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation.

    Directory of Open Access Journals (Sweden)

    Emma M Quinn

    Full Text Available Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; P(adjusted = 2.40x10(-11 in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (P(adjusted = 0.002, and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10(-16 and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; P(adjusted = 3.6x10(-3 as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10(-16 indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis.

  8. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families...

  9. Real-time imaging of resident T cells in human lung and ovarian carcinomas reveals how different tumor microenvironments control T lymphocyte migration

    Directory of Open Access Journals (Sweden)

    Houcine eBougherara

    2015-10-01

    Full Text Available T cells play a key role in the battle against cancer. To perform their antitumor activities, T cells need to adequately respond to tumor antigens by establishing contact with either malignant cells or antigen-presenting cells. These latter functions rely on a series of migratory steps that go from entry of T cells into the tumor followed by their locomotion in the tumor stroma. Our knowledge of how T cells migrate within tumors mainly comes from experiments performed in mouse models. Whereas such systems have greatly advanced our understanding, they do not always faithfully recapitulate the disease observed in cancer patients. We previously described a technique based on tissue slices that enables to track with real-time imaging microscopy the motile behavior of fluorescent T cells plated onto fresh sections of human lung tumors. We have now refined this approach to monitor the locomotion of resident tumor-infiltrating CD8 T cells labeled with fluorescently-coupled antibodies. Using this approach, our findings reveal that CD8 T cells accumulate in the stroma of ovarian and lung carcinomas but move slowly in this compartment. Conversely, even though less populated, tumors islets were found to be zones of faster migration for resident CD8 T cells. We also confirm the key role played by collagen fibers which, by their orientation, spacing and density, control the distribution and migration of resident CD8 T cells within the tumor stroma. We have subsequently demonstrated that under some physical tissue constraints CD8 T cells exhibited a mode of migration characterized by alternate forward and backward movements. In sum, using an ex vivo assay to track CD8 T cells in fresh human tumor tissues, we have identified the extracellular matrix as a major stromal component in influencing T cell migration, thereby impacting control of tumor growth. This approach will aid in the development and testing of novel immunotherapy strategies to promote T cell

  10. Spontaneous kisspeptin neuron firing in the adult mouse reveals marked sex and brain region differences but no support for a direct role in negative feedback.

    Science.gov (United States)

    de Croft, Simon; Piet, Richard; Mayer, Christian; Mai, Oliver; Boehm, Ulrich; Herbison, Allan E

    2012-11-01

    Kisspeptin-Gpr54 signaling is critical for the GnRH neuronal network controlling fertility. The present study reports on a kisspeptin (Kiss)-green fluorescent protein (GFP) mouse model enabling brain slice electrophysiological recordings to be made from Kiss neurons in the arcuate nucleus (ARN) and rostral periventricular region of the third ventricle (RP3V). Using dual immunofluorescence, approximately 90% of GFP cells in the RP3V of females, and ARN in both sexes, are shown to be authentic Kiss-synthesizing neurons in adult mice. Cell-attached recordings of ARN Kiss-GFP cells revealed a marked sex difference in their mean firing rates; 90% of Kiss-GFP cells in males exhibited slow irregular firing (0.17 ± 0.04 Hz) whereas neurons from diestrous (0.01 ± 0.01 Hz) and ovariectomized (0 Hz) mice were mostly or completely silent. In contrast, RP3V Kiss-GFP cells were all spontaneously active, exhibiting tonic, irregular, and bursting firing patterns. Mean firing rates were significantly (P neurons at the time of the proestrous GnRH surge revealed a significant decline in firing rate after the surge. Together, these observations demonstrate unexpected sex differences in the electrical activity of ARN Kiss neurons and markedly different patterns of firing by Kiss neurons in the ARN and RP3V. Although data supported a positive influence of gonadal steroids on RP3V Kiss neuron firing, no direct evidence was found to support the previously postulated role of ARN Kiss neurons in the estrogen-negative feedback mechanism.

  11. Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias.

    Science.gov (United States)

    Morgan, Suzanne M; Hodges, Elizabeth; Mitchell, Tracey J; Harris, Susan; Whittaker, Sean J; Smith, John L

    2006-08-01

    Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.

  12. The role of CD8+ T cells during allograft rejection

    Directory of Open Access Journals (Sweden)

    Bueno V.

    2002-01-01

    Full Text Available Organ transplantation can be considered as replacement therapy for patients with end-stage organ failure. The percent of one-year allograft survival has increased due, among other factors, to a better understanding of the rejection process and new immunosuppressive drugs. Immunosuppressive therapy used in transplantation prevents activation and proliferation of alloreactive T lymphocytes, although not fully preventing chronic rejection. Recognition by recipient T cells of alloantigens expressed by donor tissues initiates immune destruction of allogeneic transplants. However, there is controversy concerning the relative contribution of CD4+ and CD8+ T cells to allograft rejection. Some animal models indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas in others CD4-depleted mice reject certain types of allografts. Moreover, there is evidence that CD8+ T cells are more resistant to immunotherapy and tolerance induction protocols. An intense focal infiltration of mainly CD8+CTLA4+ T lymphocytes during kidney rejection has been described in patients. This suggests that CD8+ T cells could escape from immunosuppression and participate in the rejection process. Our group is primarily interested in the immune mechanisms involved in allograft rejection. Thus, we believe that a better understanding of the role of CD8+ T cells in allograft rejection could indicate new targets for immunotherapy in transplantation. Therefore, the objective of the present review was to focus on the role of the CD8+ T cell population in the rejection of allogeneic tissue.

  13. Viral factors reveal a role for REF/Aly in nuclear RNA stability.

    Science.gov (United States)

    Stubbs, Sarah H; Hunter, Olga V; Hoover, Ashley; Conrad, Nicholas K

    2012-04-01

    TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In Saccharomyces cerevisiae, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV noncoding polyadenylated nuclear (PAN) RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation, and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independently of their export and support a broader conservation of RNA quality control mechanisms from yeast to humans.

  14. Global analysis of SUMO chain function reveals multiple roles in chromatin regulation.

    Science.gov (United States)

    Srikumar, Tharan; Lewicki, Megan C; Costanzo, Michael; Tkach, Johnny M; van Bakel, Harm; Tsui, Kyle; Johnson, Erica S; Brown, Grant W; Andrews, Brenda J; Boone, Charles; Giaever, Guri; Nislow, Corey; Raught, Brian

    2013-04-01

    Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric "chains," but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3(allR)) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3(allR) cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a >1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast.

  15. Extensive cargo identification reveals distinct biological roles of the 12 importin pathways

    Science.gov (United States)

    Kimura, Makoto; Morinaka, Yuriko; Imai, Kenichiro; Kose, Shingo; Horton, Paul; Imamoto, Naoko

    2017-01-01

    Vast numbers of proteins are transported into and out of the nuclei by approximately 20 species of importin-β family nucleocytoplasmic transport receptors. However, the significance of the multiple parallel transport pathways that the receptors constitute is poorly understood because only limited numbers of cargo proteins have been reported. Here, we identified cargo proteins specific to the 12 species of human import receptors with a high-throughput method that employs stable isotope labeling with amino acids in cell culture, an in vitro reconstituted transport system, and quantitative mass spectrometry. The identified cargoes illuminated the manner of cargo allocation to the receptors. The redundancies of the receptors vary widely depending on the cargo protein. Cargoes of the same receptor are functionally related to one another, and the predominant protein groups in the cargo cohorts differ among the receptors. Thus, the receptors are linked to distinct biological processes by the nature of their cargoes. DOI: http://dx.doi.org/10.7554/eLife.21184.001 PMID:28117667

  16. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    Science.gov (United States)

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-08-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

  17. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    decrease in solvation free energy, harnessing the monomer solvation free energy earned during the misfolding. The second step, where a compact dimer structure is formed, is driven by direct protein-protein interactions, but again it is accompanied by an increase in solvation free energy. The increased solvation free energy of the dimer will function as the driving force to recruit another Aβ protein in the approach stage of subsequent oligomerizations. The fluctuating thermodynamics analysis of the misfolding and dimerization of the Aβ protein indicates that the interaction of the protein with surrounding water plays a critical role in protein aggregation. Such a water-centric perspective is further corroborated by demonstrating that, for a large number of Aβ mutants and mutants of other protein systems, the change in the experimental aggregation propensity upon mutation has a significant correlation with the protein solvation free energy change. We also find striking discrimination between the positively and negatively charged residues on the protein surface by surrounding water molecules, which is shown to play a crucial role in determining the protein aggregation propensity. We argue that the protein total charge dictates such striking behavior of the surrounding water molecules. Our results provide new insights for understanding and predicting the protein aggregation propensity, thereby offering novel design principles for producing aggregation-resistant proteins for biotherapeutics.

  18. Elucidation of the RamA regulon in Klebsiella pneumoniae reveals a role in LPS regulation.

    Directory of Open Access Journals (Sweden)

    Shyamasree De Majumdar

    2015-01-01

    Full Text Available Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

  19. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

  20. Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

    Science.gov (United States)

    Feldmann, Radmila; Fischer, Cornelius; Kodelja, Vitam; Behrens, Sarah; Haas, Stefan; Vingron, Martin; Timmermann, Bernd; Geikowski, Anne; Sauer, Sascha

    2013-04-01

    Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

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

    Directory of Open Access Journals (Sweden)

    Yue Hu

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

  2. Sarcomere dynamics in single myocardial cells as revealed by high-resolution light diffractometry.

    Science.gov (United States)

    Leung, A F

    1983-08-01

    A specially designed diffractometer with a high spatial and temporal resolution recorded the diffraction of a laser beam by single enzymatically isolated myocardial cells. The fine structures within the first-order diffraction were resolved and each structure was interpreted as the diffraction from a group of sarcomeres of nearly equal length. During activation of the cell dynamics of each discrete group of sarcomeres was uniform and independent of the other groups. However, a small nonuniform component in the sarcomere dynamics was observed and attributed to the coupling between the shortening tension and the radial stress resulting from the expansion of the myofibrillar cross-section. The time-course of the diffraction fine structures during contractile activity revealed (1) the period of the contraction-relaxation cycle, (2) the latent period, (3) the shortening and relengthening speeds and (4) the variation in the line width and intensity of the fine structure. Measurements showed that the latent period was dependent on the free Ca2+ of the cell's bathing solution while the initial shortening speed was not. The diffraction line width and intensity of the shortening cell were explained by the grating model.

  3. The role of root border cells in plant defense.

    Science.gov (United States)

    Hawes, M C; Gunawardena, U; Miyasaka, S; Zhao, X

    2000-03-01

    The survival of a plant depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Perhaps because of the root tip's vital role in plant health, it is ensheathed by large populations of detached somatic cells - root 'border' cells - which have the ability to engineer the chemical and physical properties of the external environment. Of particular significance, is the production by border cells of specific chemicals that can dramatically alter the behavior of populations of soilborne microflora. Molecular approaches are being used to identify and manipulate the expression of plant genes that control the production and the specialized properties of border cells in transgenic plants. Such plants can be used to test the hypothesis that these unusual cells act as a phalanx of biological 'goalies', which neutralize dangers to newly generated root tissue as the root tip makes its way through soil.

  4. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

    Wewer, U M; Taraboletti, G; Sobel, M E;

    1987-01-01

    Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986......). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor...... antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin...

  5. [Role of Langerhans cells in the physiopathology of atopic dermatitis].

    Science.gov (United States)

    Bieber, T

    1995-12-01

    The demonstration of IgE receptors on the surface of epidermal dendritic cells and on other antigen presenting cells is a crucial element in the understanding of the pathophysiological role of these cells in the genesis of atopic disease, and especially the atopic dermatitis (AD). The sensibilisation phase to an aeroallergen at the level of nasal or bronchial mucosa and even at the skin may be mediated by dendritic cells expressing Fc epsilon RI. Distinct forms of AD may then represent the equivalent of the ellicitation phase of the classical allergic contact dermatitis. Fc epsilon RI would lead, via specific IgE, to an efficient antigen capture, to the activation of the dendritic cells and finally to an antigen presentation. Thus, AD may represent the paradigma of an IgE-mediated type IV reaction.

  6. Role of stem cells in spondyloarthritis: Pathogenesis, treatment and complications.

    Science.gov (United States)

    Wong, Rebecca S Y

    2015-10-01

    Spondyloarthritis (SpA) is a family of interrelated inflammatory arthritis that includes ankylosing spondylitis (AS), psoriatic arthritis, reactive arthritis, arthritis related to inflammatory bowel disease and undifferentiated SpA. The classification, epidemiology, pathogenesis and treatment of SpA have been extensively reviewed in the published literature. Reviews on the use of stem cells in various autoimmune diseases in general are also common. However, a review on the role of stem cells in SpA is currently lacking. This review focuses on the involvement of stem cells in the pathogenesis of SpA and the application of different types of stem cells in the treatment of SpA. It also addresses some of the complications which may arise as a result of the use of stem cells in the treatment of SpA.

  7. Mammary development and breast cancer: the role of stem cells.

    Science.gov (United States)

    Ercan, C; van Diest, P J; Vooijs, M

    2011-06-01

    The mammary gland is a highly regenerative organ that can undergo multiple cycles of proliferation, lactation and involution, a process controlled by stem cells. The last decade much progress has been made in the identification of signaling pathways that function in these stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. The same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer. Here we provide an overview on the functional and molecular identification of mammary stem cells in the context of both normal breast development and breast cancer. We discuss the contribution of some key signaling pathways with an emphasis on Notch receptor signaling, a cell fate determination pathway often deregulated in breast cancer. A further understanding of the biological roles of the Notch pathway in mammary stem cell behavior and carcinogenesis might be relevant for the development of future therapies.

  8. Systems Chemo-Biology and Transcriptomic Meta-Analysis Reveal the Molecular Roles of Bioactive Lipids in Cardiomyocyte Differentiation.

    Science.gov (United States)

    de Faria Poloni, Joice; Bonatto, Diego

    2015-09-01

    Lipids, which are essential constituents of biological membranes, play structural and functional roles in the cell. In recent years, certain lipids have been identified as regulatory signaling molecules and have been termed "bioactive lipids". Subsequently, the importance of bioactive lipids in stem cell differentiation and cardiogenesis has gained increasing recognition. Therefore, the aim of this study was to identify the biological processes underlying murine cardiac differentiation and the mechanisms by which bioactive lipids affect these processes. For this purpose, a transcriptomic meta-analysis of microarray and RNA-seq data from murine stem cells undergoing cardiogenic differentiation was performed. The differentially expressed genes identified via this meta-analysis, as well as bioactive lipids, were evaluated using systems chemo-biology tools. These data indicated that bioactive lipids are associated with the regulation of cell motility, cell adhesion, cytoskeletal rearrangement, and gene expression. Moreover, bioactive lipids integrate the signaling pathways involved in cell migration, the secretion and remodeling of extracellular matrix components, and the establishment of the cardiac phenotype. In conclusion, this study provides new insights into the contribution of bioactive lipids to the induction of cellular responses to various stimuli, which may originate from the extracellular environment and morphogens, and the manner in which this contribution directly affects murine heart morphogenesis.

  9. A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

    Directory of Open Access Journals (Sweden)

    Ryan Chao

    Full Text Available Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

  10. A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

    Science.gov (United States)

    Chao, Ryan; Nevin, Linda; Agarwal, Pooja; Riemer, Jan; Bai, Xiaoyang; Delaney, Allen; Akana, Matthew; JimenezLopez, Nelson; Bardakjian, Tanya; Schneider, Adele; Chassaing, Nicolas; Schorderet, Daniel F; FitzPatrick, David; Kwok, Pui-yan; Ellgaard, Lars; Gould, Douglas B; Zhang, Yan; Malicki, Jarema; Baier, Herwig; Slavotinek, Anne

    2010-05-11

    Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

  11. The role of starburst amacrine cells in visual signal processing

    Science.gov (United States)

    TAYLOR, W.R.; SMITH, R.G.

    2012-01-01

    Starburst amacrine cells (SBACs) within the adult mammalian retina provide the critical inhibition that underlies the receptive field properties of direction-selective ganglion cells (DSGCs). The SBACs generate direction-selective output of GABA that differentially inhibits the DSGCs. We review the biophysical mechanisms that produce directional GABA release from SBACs and test a network model that predicts the effects of reciprocal inhibition between adjacent SBACs. The results of the model simulations suggest that reciprocal inhibitory connections between closely spaced SBACs should be spatially selective, while connections between more widely spaced cells could be indiscriminate. SBACs were initially identified as cholinergic neurons and were subsequently shown to contain release both acetylcholine and GABA. While the role of the GABAergic transmission is well established, the role of the cholinergic transmission remains unclear. PMID:22310373

  12. A novel meta-analysis approach of cancer transcriptomes reveals prevailing transcriptional networks in cancer cells.

    Science.gov (United States)

    Niida, Atsushi; Imoto, Seiya; Nagasaki, Masao; Yamaguchi, Rui; Miyano, Satoru

    2010-01-01

    Although microarray technology has revealed transcriptomic diversities underlining various cancer phenotypes, transcriptional programs controlling them have not been well elucidated. To decode transcriptional programs governing cancer transcriptomes, we have recently developed a computational method termed EEM, which searches for expression modules from prescribed gene sets defined by prior biological knowledge like TF binding motifs. In this paper, we extend our EEM approach to predict cancer transcriptional networks. Starting from functional TF binding motifs and expression modules identified by EEM, we predict cancer transcriptional networks containing regulatory TFs, associated GO terms, and interactions between TF binding motifs. To systematically analyze transcriptional programs in broad types of cancer, we applied our EEM-based network prediction method to 122 microarray datasets collected from public databases. The data sets contain about 15000 experiments for tumor samples of various tissue origins including breast, colon, lung etc. This EEM based meta-analysis successfully revealed a prevailing cancer transcriptional network which functions in a large fraction of cancer transcriptomes; they include cell-cycle and immune related sub-networks. This study demonstrates broad applicability of EEM, and opens a way to comprehensive understanding of transcriptional networks in cancer cells.

  13. Profiling of microRNA-mRNA reveals roles of microRNAs in cervical cancer

    Institute of Scientific and Technical Information of China (English)

    MA Ding; ZHANG You-yi; GUO Yan-li; LI Zi-jian; GENG Li

    2012-01-01

    Background Cervical cancer is one of the most common malignant tumors in women.This study was designed to explore the expression profiles of microRNAs (miRNAs) and mRNAs and the gene regulation network in cervical tumorigenesis and to find candidate molecular markers and key tumorigenic genes in cervical cancer.Methods miRNAs and mRNAs expression microarrays were used to detect the expression of miRNAs and mRNAs in normal and cancer cervical tissues.TargetScan 5.0 database (UK) was used to predict the target genes of the miRNAs,analyze their intersection with differentially expressed mRNAs and negatively correlate the intersection with miRNAs.Bioinformatic approaches were used to analyze functions and pathways of the target genes and establish miRNA-gene network.Results Twenty-nine miRNAs and 2036 mRNAs were differentially expressed in normal and cervical tumor tissues.Among them,13 miRNAs and 754 mRNAs were up-regulated in cervical tumor tissues and 16 miRNAs and 1282 RNA were down-regulated.The 327 target genes negatively related to miRNAs in the intersection were involved in functions and signal pathways.Down-regulated miRNAs targeted genes and up-regulated miRNAs targeted genes were involved in 415 and 163 functions,respectively,and in 37 and 17 significant pathways,respectively (P <0.05,false discovery rate (FDR) <0.05).We constructed the miRNAs-gene network and found that hsa-miR-15a,hsa-miR-106b and hsa-miR-20b were key nodes in the network.Conclusions The differentially expressed miRNAs and mRNAs in cervical cancer and related miRNA-gene network have been identified.They play important roles in cervical tumorigenesis and are involved in many important biological functions and signal transduction pathways.These findings lay a foundation for research on the molecular mechanism of miRNAs in the pathogenesis of cervical cancer.

  14. Functional profiles reveal unique ecological roles of various biological soil crust organisms

    Science.gov (United States)

    Bowker, M.A.; Mau, R.L.; Maestre, F.T.; Escolar, C.; Castillo-Monroy, A. P.

    2011-01-01

    1. At the heart of the body of research on biodiversity effects on ecosystem function is the debate over whether different species tend to be functionally singular or redundant. When we consider ecosystem multi-function, the provision of multiple ecosystem functions simultaneously, we may find that seemingly redundant species may in fact play unique roles in ecosystems. 2. Over the last few decades, the significance of biological soil crusts (BSCs) as ecological boundaries and ecosystem engineers, and their multi-functional nature, has become increasingly well documented. We compiled 'functional profiles' of the organisms in this understudied community, to determine whether functional singularity emerges when multiple ecosystem functions are considered. 3. In two data sets, one representing multiple sites around the semi-arid regions of Spain (regional scale), and another from a single site in central Spain (local scale), we examined correlations between the abundance or frequency of BSC species in a community, and multiple surrogates of ecosystem functioning. There was a wide array of apparent effects of species on specific functions. 4. Notably, in gypsiferous soils and at regional scale, we found that indicators of carbon (C) and phosphorus cycling were apparently suppressed and promoted by the lichens Diploschistes diacapsis and Squamarina lentigera, respectively. The moss Pleurochaete squarrosa appears to promote C cycling in calcareous soils at this spatial scale. At the local scale in gypsiferous soils, D. diacapsis positively correlated with carbon cycling, but negatively with nitrogen cycling, whereas numerous lichens exhibited the opposite profile. 5. We found a high degree of functional singularity, i.e. that species were highly individualistic in their effects on multiple functions. Many functional attributes were not easily predictable from existing functional grouping systems based primarily on morphology. 6. Our results suggest that maintaining

  15. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  16. Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells

    DEFF Research Database (Denmark)

    Lauber, Chris; Vieyres, Gabrielle; Terczynska-Dyla, Ewa

    2015-01-01

    The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IF...... genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium....

  17. Root border-like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria.

    Science.gov (United States)

    Vicré, Maïté; Santaella, Catherine; Blanchet, Sandrine; Gateau, Aurélien; Driouich, Azeddine

    2005-06-01

    Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.

  18. Langerhans cells and their role in oral mucosal diseases

    Directory of Open Access Journals (Sweden)

    Juhi Upadhyay

    2013-01-01

    Full Text Available Dendritic cells are arguably the most potent antigen-presenting cells and may be the only cells capable of initiating the adaptive immune response. The epithelial residents of dendritic cells are Langerhans cells, which serve as the "sentinels" of the mucosa, altering the immune system not only to pathogen entry but also of tolerance to self antigen and commensal microbes. Oral mucosal Langerhans cells are capable of engaging and internalizing a wide variety of pathogens and have been found responsive to nickel in patients with nickel allergies, oral Candida species, oral lichen planus, lichenoid drug eruptions, graft versus host diseases, periodontal diseases median rhomboid glossitis, human immunodeficiency virus infection, hairy leukoplakia of the tongue, and oral squamous cell carcinoma. Review focuses on the role of antigen-presenting cells in particular Langerhans cells to better understand the mechanisms underlying immune responses. In this review, comprehensive detail about mucosal diseases has been compiled using the PubMed database and through textbooks.

  19. The roles and regulation of Sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis.

    Science.gov (United States)

    Hai, Yanan; Hou, Jingmei; Liu, Yun; Liu, Yang; Yang, Hao; Li, Zheng; He, Zuping

    2014-05-01

    Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs) self-renew and differentiate into spermatozoa under the elaborate coordination of testicular microenvironment, namely, niche. Sertoli cells, which locate around male germ cells, are the most critical component of the niche. Significant progress has recently been made by peers and us on uncovering the effects of Sertoli cells on regulating fate determinations of SSCs. Here we addressed the roles and regulation of Sertoli cells in normal and abnormal spermatogenesis. Specifically, we summarized the biological characteristics of Sertoli cells, and we emphasized the roles of Sertoli cells in mediating the self-renewal, differentiation, apoptosis, de-differentiation, and trans-differentiation of SSCs. The association between abnormal function of Sertoli cells and impaired spermatogenesis was discussed. Finally, we highlighted several issues to be addressed for further investigation on the effects and mechanisms of Sertoli cells in spermatogenesis. Since Sertoli cells are the key supportive cells for SSCs and they are very receptive to modification, a better understanding of the roles and regulation of Sertoli cells in SSC biology and spermatogenesis would make it feasible to identify novel targets for gene therapy of male infertility as well as seek more efficient and safer strategies for male contraception.

  20. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

    Directory of Open Access Journals (Sweden)

    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  1. Exosomes secreted from human colon cancer cells influence the adhesion of neighboring metastatic cells: Role of microRNA-210

    Science.gov (United States)

    Bigagli, Elisabetta; Luceri, Cristina; Guasti, Daniele; Cinci, Lorenzo

    2016-01-01

    ABSTRACT Cancer-secreted exosomes influence tumor microenvironment and support cancer growth and metastasis. MiR-210 is frequently up-regulated in colorectal cancer tissues and correlates with metastatic disease. We investigated whether exosomes are actively released by HCT-8 colon cancer cells, the role of exosomal miR-210 in the cross-talk between primary cancer cells and neighboring metastatic cells and its contribution in regulating epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). After 7 d of culture, a subpopulation of viable HCT-8 cells detached the monolayer and started to grow in suspension, suggesting anoikis resistance and a metastatic potential. The expression of key proteins of EMT revealed that these cells were E-cadherin negative and vimentin positive further confirming their metastatic phenotype and the acquisition of anoikis resistance. Metastatic cells, in the presence of adherently growing HCT-8, continued to grow in suspension whereas only if seeded in cell-free wells, were able to adhere again and to form E-cadherin positive and vimentin negative new colonies, suggesting the occurrence of MET. The chemosensitivity to 5 fluorouracil and to FOLFOX-like treatment of metastatic cells was significantly diminished compared to adherent HCT-8 cells. Of note, adherent new colonies undergoing MET, were insensitive to both chemotherapeutic strategies. Electron microscopy analysis demonstrated that adherently growing HCT-8, actually secreted exosomes and that exosomes in turn were taken up by metastatic cells. When exosomes secreted by adherently growing HCT-8 were administered to metastatic cells, MET was significantly inhibited. miR-210 was significantly upregulated in exosomes compared to its intracellular levels in adherently growing HCT-8 cells and correlated to anoikis resistance and EMT markers. Exosomes containing miR-210 might be considered as EMT promoting signals that preserve the local cancer

  2. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Keiko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute of Life Sciences for the Next Generation of Women Scientists, Fukuoka University, Fukuoka (Japan); Fujimoto, Takahiro [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Okamura, Tadashi [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ogawa, Masahiro [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tanaka, Yoko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Mototani, Yasumasa; Goto, Motohito [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ota, Takeharu; Matsuzaki, Hiroshi [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Kuroki, Masahide [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tsunoda, Toshiyuki [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Sasazuki, Takehiko [Institute for Advanced Study, Kyushu University, Fukuoka (Japan); Shirasawa, Senji, E-mail: sshirasa@fukuoka-u.ac.jp [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  3. Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions.

    Directory of Open Access Journals (Sweden)

    Matthew L Katz

    Full Text Available Sensory stimuli are encoded by diverse kinds of neurons but the identities of the recorded neurons that are studied are often unknown. We explored in detail the firing patterns of eight previously defined genetically-identified retinal ganglion cell (RGC types from a single transgenic mouse line. We first introduce a new technique of deriving receptive field vectors (RFVs which utilises a modified form of mutual information ("Quadratic Mutual Information". We analysed the firing patterns of RGCs during presentation of short duration (~10 second complex visual scenes (natural movies. We probed the high dimensional space formed by the visual input for a much smaller dimensional subspace of RFVs that give the most information about the response of each cell. The new technique is very efficient and fast and the derivation of novel types of RFVs formed by the natural scene visual input was possible even with limited numbers of spikes per cell. This approach enabled us to estimate the 'visual memory' of each cell type and the corresponding receptive field area by calculating Mutual Information as a function of the number of frames and radius. Finally, we made predictions of biologically relevant functions based on the RFVs of each cell type. RGC class analysis was complemented with results for the cells' response to simple visual input in the form of black and white spot stimulation, and their classification on several key physiological metrics. Thus RFVs lead to predictions of biological roles based on limited data and facilitate analysis of sensory-evoked spiking data from defined cell types.

  4. Molecular characterization of Helicobacter pylori VacA induction of IL-8 in U937 cells reveals a prominent role for p38MAPK in activating transcription factor-2, cAMP response element binding protein, and NF-kappaB activation

    DEFF Research Database (Denmark)

    Hisatsune, Junzo; Nakayama, Masaaki; Isomoto, Hajime;

    2008-01-01

    Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechan......Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined...... the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IkappaBalpha ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca(2+) entry (SKF96365......+) in mediating activation of MAPK and the canonical NF-kappaB pathway. VacA stimulated translocation of NF-kappaBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-kappaB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which...

  5. Potential primary roles of glial cells in the mechanisms of psychiatric disorders.

    Science.gov (United States)

    Yamamuro, Kazuhiko; Kimoto, Sohei; Rosen, Kenneth M; Kishimoto, Toshifumi; Makinodan, Manabu

    2015-01-01

    While neurons have long been considered the major player in multiple brain functions such as perception, emotion, and memory, glial cells have been relegated to a far lesser position, acting as merely a "glue" to support neurons. Multiple lines of recent evidence, however, have revealed that glial cells such as oligodendrocytes, astrocytes, and microglia, substantially impact on neuronal function and activities and are significantly involved in the underlying pathobiology of psychiatric disorders. Indeed, a growing body of evidence indicates that glial cells interact extensively with neurons both chemically (e.g., through neurotransmitters, neurotrophic factors, and cytokines) and physically (e.g., through gap junctions), supporting a role for these cells as likely significant modifiers not only of neural function in brain development but also disease pathobiology. Since questions have lingered as to whether glial dysfunction plays a primary role in the biology of neuropsychiatric disorders or a role related solely to their support of neuronal physiology in these diseases, informative and predictive animal models have been developed over the last decade. In this article, we review recent findings uncovered using glia-specific genetically modified mice with which we can evaluate both the causation of glia dysfunction and its potential role in neuropsychiatric disorders such as autism and schizophrenia.

  6. Cardiac Metastases of Renal Cell Carcinoma Revealed by Syncope: Diagnosis and Treatment

    Directory of Open Access Journals (Sweden)

    Aziz Bazine

    2014-08-01

    Full Text Available Introduction: Cardiac metastases from renal cell carcinoma are very rare. In this report, we describe a case of ventricular metastases in the absence of vena cava or right atrial involvement. Case Report: We report the case of a 60-year-old man who had a past history of heavy tobacco intake and well-controlled arterial hypertension. He experienced sudden-onset palpitations, lost consciousness and, as a result, was involved in an accident on the public highway. Cardiac arrhythmia was suspected and, therefore, transthoracic echocardiography was suggested, which revealed a large right ventricular mass. Chest and abdominal computed tomography demonstrated a mass in the right ventricle, but without contiguous vena cava involvement, and a right renal mass related to the probable neoplasm. An ultrasound-guided renal biopsy showed a clear-cell renal cell carcinoma. A bone scan revealed a metastatic bone disease. The patient was started on sunitinib treatment, which was well tolerated. However, approximately 8 months later, reevaluation showed pulmonary metastases. The patient was subsequently started on treatment with everolimus, which, however, was poorly tolerated. Two months later, the patient died due to terminal respiratory insufficiency. Discussion: Based on the literature and our observations in this case, targeted antiangiogenic therapy should be considered as a viable therapeutic alternative to metastasectomy for patients with inoperable cardiac metastatic disease as long as there is no baseline systolic or diastolic dysfunction. The case also emphasizes the importance of a thorough history review and physical examination in the workup of patients with syncope.

  7. Role of MXD3 in proliferation of DAOY human medulloblastoma cells.

    Directory of Open Access Journals (Sweden)

    Gustavo A Barisone

    Full Text Available A subset of medulloblastomas, the most common brain tumor in children, is hypothesized to originate from granule neuron precursors (GNPs in which the sonic hedgehog (SHH pathway is over-activated. MXD3, a basic helix-look-helix zipper transcription factor of the MAD family, has been reported to be upregulated during postnatal cerebellar development and to promote GNP proliferation and MYCN expression. Mxd3 is upregulated in mouse models of medulloblastoma as well as in human medulloblastomas. Therefore, we hypothesize that MXD3 plays a role in the cellular events that lead to medulloblastoma biogenesis. In agreement with its proliferative role in GNPs, MXD3 knock-down in DAOY cells resulted in decreased proliferation. Sustained overexpression of MXD3 resulted in decreased cell numbers due to increased apoptosis and cell cycle arrest. Structure-function analysis revealed that the Sin3 interacting domain, the basic domain, and binding to E-boxes are essential for this activity. Microarray-based expression analysis indicated up-regulation of 84 genes and down-regulation of 47 genes. Potential direct MXD3 target genes were identified by ChIP-chip. Our results suggest that MXD3 is necessary for DAOY medulloblastoma cell proliferation. However, increased level and/or duration of MXD3 expression ultimately reduces cell numbers via increased cell death and cell cycle arrest.

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

    Science.gov (United States)

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

    2016-02-16

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

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

    Science.gov (United States)

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

    2015-06-19

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

  10. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    OpenAIRE

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed ...

  11. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

    Directory of Open Access Journals (Sweden)

    Kelsi M Sandoz

    Full Text Available A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV and small cell variant (SCV forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV, 5 (late LCV, 7 (intermediate forms, 14 (early SCV, and 21 days (late SCV post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG, a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.

  12. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

    Science.gov (United States)

    Sandoz, Kelsi M; Popham, David L; Beare, Paul A; Sturdevant, Daniel E; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.

  13. Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila

    Science.gov (United States)

    Bailey, Andrew P.; Koster, Grielof; Guillermier, Christelle; Hirst, Elizabeth M.A.; MacRae, James I.; Lechene, Claude P.; Postle, Anthony D.; Gould, Alex P.

    2015-01-01

    Summary Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts. PMID:26451484

  14. A novel Atoh1 "self-terminating" mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability.

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    Ning Pan

    Full Text Available Atonal homolog1 (Atoh1 is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO mouse line using Tg(Atoh1-cre, in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

  15. Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing

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    Muammar eMansor

    2015-08-01

    Full Text Available Large, sulfur-cycling, calcite-precipitating bacteria in the genus Achromatium represent a significant proportion of bacterial communities near sediment-water interfaces throughout the world. Our understanding of their potentially crucial roles in calcium, carbon, sulfur, nitrogen, and iron cycling is limited because they have not been cultured or sequenced using environmental genomics approaches to date. We utilized single-cell genomic sequencing to obtain one incomplete and two nearly complete draft genomes for Achromatium collected at Warm Mineral Springs, FL. Based on 16S rRNA gene sequences, the three cells represent distinct and relatively distant Achromatium populations (91-92% identity. The draft genomes encode key genes involved in sulfur and hydrogen oxidation; oxygen, nitrogen and polysulfide respiration; carbon and nitrogen fixation; organic carbon assimilation and storage; chemotaxis; twitching motility; antibiotic resistance; and membrane transport. Known genes for iron and manganese energy metabolism were not detected. The presence of pyrophosphatase and vacuolar (V-type ATPases, which are generally rare in bacterial genomes, suggests a role for these enzymes in calcium transport, proton pumping, and/or energy generation in the membranes of calcite-containing inclusions.

  16. Novel hypophysiotropic AgRP2 neurons and pineal cells revealed by BAC transgenesis in zebrafish

    Science.gov (United States)

    Shainer, Inbal; Buchshtab, Adi; Hawkins, Thomas A.; Wilson, Stephen W.; Cone, Roger D.; Gothilf, Yoav

    2017-01-01

    The neuropeptide agouti-related protein (AgRP) is expressed in the arcuate nucleus of the mammalian hypothalamus and plays a key role in regulating food consumption and energy homeostasis. Fish express two agrp genes in the brain: agrp1, considered functionally homologous with the mammalian AgRP, and agrp2. The role of agrp2 and its relationship to agrp1 are not fully understood. Utilizing BAC transgenesis, we generated transgenic zebrafish in which agrp1- and agrp2-expressing cells can be visualized and manipulated. By characterizing these transgenic lines, we showed that agrp1-expressing neurons are located in the ventral periventricular hypothalamus (the equivalent of the mammalian arcuate nucleus), projecting throughout the hypothalamus and towards the preoptic area. The agrp2 gene was expressed in the pineal gland in a previously uncharacterized subgroup of cells. Additionally, agrp2 was expressed in a small group of neurons in the preoptic area that project directly towards the pituitary and form an interface with the pituitary vasculature, suggesting that preoptic AgRP2 neurons are hypophysiotropic. We showed that direct synaptic connection can exist between AgRP1 and AgRP2 neurons in the hypothalamus, suggesting communication and coordination between AgRP1 and AgRP2 neurons and, therefore, probably also between the processes they regulate. PMID:28317906

  17. Alcoholic hepatitis: The pivotal role of Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Duminda; B; Suraweera; Ashley; N; Weeratunga; Robert; W; Hu; Stephen; J; Pandol; Richard; Hu

    2015-01-01

    Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis(AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides(LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called tolllike receptor 4. This endotoxin mediated activation of Kupffer cells plays an important role in the inflammatory process resulting in alcoholic hepatitis. There is no effective treatment for AH, although notable progress has been made over the last decade in understanding the underlying mechanism of alcoholic hepatitis. We specifically review the current research on the role of Kupffer cells in the pathogenesis of AH and the treatment strategies. We suggest that the imbalance between the pro-inflammatory and the anti-inflammatory process as well as the increased production of reactive oxygen species eventually lead to hepatocyte injury, the final event of alcoholic hepatitis.

  18. Roles for microtubule and microfilament cytoskeletons in animal cell cytokinesis

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhongcai; CAI Shang; JIANG Qing; ZHANG Chuanmao; TANG Xiaowei

    2005-01-01

    Microtubule and microfilament cytoskeletons play key roles in the whole process of cytokinesis. Although a number of hypotheses have been proposed to elucidate the mechanism of cytokinesis by microtubule and actin filament cytoskeletons, many reports are conflicting. In our study, combining the cytoskeletons drug treatments with the time-lapse video technology, we retested the key roles of microtubule and actin filament in cytokinesis. The results showed that depolymerization of microtubules by Nocodazole after the initiation of furrowing would not inhibit the furrow ingression, but obviously decrease the stiffness of daughter cells. Depolymerizing actin filaments by Cytochalasin B before metaphase would inhibit the initiation of furrowing but not chromosome segregation, resulting in the formation of binucleate cells; however, depolymerizing actin filaments during anaphase would prevent furrowing and lead to the regress of established furrow, also resulting in the formation of binucleate cells. Further, depolymerizing microtubules and actin filaments simultaneously after metaphase would cause the quick regress of the furrow and the formation of binucleate cells. From these results we propose that a successful cytokinesis requires functions and coordination of both the microtubule and actin filament cytoskeletons. Microtubule cytoskeleton may function in the positioning and initiation of cleavage furrow, and the actin filament cytoskeleton may play key roles in the initiation and ingression of the furrow.

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

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    Daniel J Anderson

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

  20. Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.

    Science.gov (United States)

    Bischofberger, Mirko; Iacovache, Ioan; Boss, Daniel; Naef, Felix; van der Goot, F Gisou; Molina, Nacho

    2016-04-12

    Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.

  1. Live imaging reveals a new role for the sigma-1 (σ1) receptor in allowing microglia to leave brain injuries.

    Science.gov (United States)

    Moritz, Christian; Berardi, Francesco; Abate, Carmen; Peri, Francesca

    2015-03-30

    Microglial cells are responsible for clearing and maintaining the central nervous system (CNS) microenvironment. Upon brain damage, they move toward injuries to clear the area by engulfing dying neurons. However, in the context of many neurological disorders chronic microglial responses are responsible for neurodegeneration. Therefore, it is important to understand how these cells can be "switched-off" and regain their ramified state. Current research suggests that microglial inflammatory responses can be inhibited by sigma (σ) receptor activation. Here, we take advantage of the optical transparency of the zebrafish embryo to study the role of σ1 receptor in microglia in an intact living brain. By combining chemical approaches with real time imaging we found that treatment with PB190, a σ1 agonist, blocks microglial migration toward injuries leaving cellular baseline motility and the engulfment of apoptotic neurons unaffected. Most importantly, by taking a reverse genetic approach, we discovered that the role of σ1in vivo is to "switch-off" microglia after they responded to an injury allowing for these cells to leave the site of damage. This indicates that pharmacological manipulation of σ1 receptor modulates microglial responses providing new approaches to reduce the devastating impact that microglia have in neurodegenerative diseases.

  2. Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation.

    Science.gov (United States)

    Naylor, Matthew J; Li, Na; Cheung, Julia; Lowe, Emma T; Lambert, Elise; Marlow, Rebecca; Wang, Pengbo; Schatzmann, Franziska; Wintermantel, Timothy; Schüetz, Günther; Clarke, Alan R; Mueller, Ulrich; Hynes, Nancy E; Streuli, Charles H

    2005-11-21

    Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.

  3. Role of leptin receptors in granulosa cells during ovulation.

    Science.gov (United States)

    Dupuis, Lisa; Schuermann, Yasmin; Cohen, Tamara; Siddappa, Dayananda; Kalaiselvanraja, Anitha; Pansera, Melissa; Bordignon, Vilceu; Duggavathi, Raj

    2014-02-01

    Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein β (Cebpβ) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpβ to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpβ, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 μg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpβ. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

  4. Role of Dicer on tumorigenesis in glioma cells

    Institute of Scientific and Technical Information of China (English)

    Anling Zhang; Lei Han; Guangxiu Wang; Zhifan Jia; Peiyu Pu; Chunsheng Kang

    2010-01-01

    Micro RNAs(miRNAs)are non-coding,single-stranded RNAs that regulate target gene expression by repressing translation or promoting RNA cleavage.Recent studies show that miRNA expression is globally decreased in some human tumors.Dicer is an essential component of the miRNA processing machinery.To determine whether global reduction of miRNA effects tumorigenesis,small interfering RNA were designed to target Dicer to restrain whole miRNA expression in the glioblastoma cell line-TJ905.With effective knock-down of Dicer,tumor cells were invasive and proliferative,and globally impaired miRNA processing enhanced proliferation and invasiveness of glioma cells in vitro.Suppression of Dicer expression resulted in a more aggressive glioma phenotype,which suggests that global reduction of miRNA expression could have an oncogenic role in glioblastoma cells.

  5. Role of Ikaros in T-cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    Philippe; Kastner; Susan; Chan

    2011-01-01

    Ikaros is a zinc finger transcriptional regulator encoded by the Ikzf1 gene.Ikaros displays crucial functions in the hematopoietic system and its loss of function has been linked to the development of lymphoid leukemia.In particular,Ikaros has been found in recent years to be a major tumor suppressor involved in human B-cell acute lymphoblastic leukemia.Its role in T-cell leukemia,however,has been more controversial.While Ikaros deficiency appears to be very frequent in murine T-cell leukemias,loss of Ikaros appears to be rare in human T-cell acute lymphoblastic leukemia (T-ALL).We review here the evidence linking Ikaros to T-ALL in mouse and human systems.

  6. Sperm associated antigen 9 plays an important role in bladder transitional cell carcinoma.

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    Deepika Kanojia

    Full Text Available BACKGROUND: Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9 in bladder TCC. METHODOLOGY AND FINDINGS: We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042 and low grade tumors (P = 0.002 suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G0-G1 arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro. CONCLUSIONS: Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.

  7. Cell-intrinsic role for NF-kappa B-inducing kinase in peripheral maintenance but not thymic development of Foxp3+ regulatory T cells in mice.

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    Susan E Murray

    Full Text Available NF-κB inducing kinase (NIK, MAP3K14 is a key signaling molecule in non-canonical NF-κB activation, and NIK deficient mice have been instrumental in deciphering the immunologic role of this pathway. Global ablation of NIK prevents lymph node development, impairs thymic stromal development, and drastically reduces B cells. Despite altered thymic selection, T cell numbers are near normal in NIK deficient mice. The exception is CD4(+ regulatory T cells (Tregs, which are reduced in the thymus and periphery. Defects in thymic stroma are known to contribute to impaired Treg generation, but whether NIK also plays a cell intrinsic role in Tregs is unknown. Here, we compared intact mice with single and mixed BM chimeric mice to assess the intrinsic role of NIK in Treg generation and maintenance. We found that while NIK expression in stromal cells suffices for normal thymic Treg development, NIK is required cell-intrinsically to maintain peripheral Tregs. In addition, we unexpectedly discovered a cell-intrinsic role for NIK in memory phenotype conventional T cells that is masked in intact mice, but revealed in BM chimeras. These results demonstrate a novel role for NIK in peripheral regulatory and memory phenotype T cell homeostasis.

  8. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome

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    Frédéric Pontvianne

    2016-08-01

    Full Text Available The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS and identified nucleolus-associated chromatin domains (NADs by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1. NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs, sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  9. The role of fibrocytes in sickle cell lung disease.

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    Joshua J Field

    Full Text Available BACKGROUND: Interstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease. METHODOLOGY/PRINCIPAL FINDINGS: Fibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle