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Sample records for cells reveals roles

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

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    Masato S Ota

    2009-04-01

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

  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. Targeted deletion of p73 in mice reveals its role in T cell development and lymphomagenesis.

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    Alice Nemajerova

    Full Text Available Transcriptional silencing of the p73 gene through methylation has been demonstrated in human leukemias and lymphomas. However, the role of p73 in the malignant process remains to be explored. We show here that p73 acts as a T cell-specific tumor suppressor in a genetically defined mouse model, and that concomitant ablation of p53 and p73 predisposes mice to an increased incidence of thymic lymphomas compared to the loss of p53 alone. Our results demonstrate a causal role for loss of p73 in progression of T cell lymphomas to the stage of aggressive, disseminated disease. We provide evidence that tumorigenesis in mice lacking p53 and p73 proceeds through mechanisms involving altered patterns of gene expression, defects in early T cell development, impaired apoptosis, and the ensuing accumulation of chromosomal aberrations. Collectively, our data imply that tumor suppressive properties of p73 are highly dependent on cellular context, wherein p73 plays a major role in T cell development and neoplasia.

  4. Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

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    Calcagnì, Alessia; kors, Lotte; Verschuren, Eric; De Cegli, Rossella; Zampelli, Nicolina; Nusco, Edoardo; Confalonieri, Stefano; Bertalot, Giovanni; Pece, Salvatore; Settembre, Carmine; Malouf, Gabriel G; Leemans, Jaklien C; de Heer, Emile; Salvatore, Marco; Peters, Dorien JM; Di Fiore, Pier Paolo; Ballabio, Andrea

    2016-01-01

    TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway. DOI: http://dx.doi.org/10.7554/eLife.17047.001

  5. 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.

  6. Stem cell heterogeneity revealed

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    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....

  7. Combined In Silico and In Vivo Analyses Reveal Role of Hes1 in Taste Cell Differentiation

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    Ota, Masato S.; Yoshiyuki Kaneko; Kaori Kondo; Soichi Ogishima; Hiroshi Tanaka; Kazuhiro Eto; Takashi Kondo

    2009-01-01

    Author Summary The sensation of taste is composed of five basic modalities: sweet, bitter, umami, sour, and salty. Specialized taste cells perceive the various chemical cues within food. About 100 taste cells assemble into onion-shaped clusters called taste buds, which are located on taste papillae in the tongue epithelium and on oral mucosa. Of the five taste modalities, the taste stimulants responsible for sweet, bitter, and umami tastes are recognized by a group of G protein–coupled taste ...

  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. Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

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    Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2016-01-01

    In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti

  10. 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.

  11. 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

  12. Role of astroglia in down's syndrome revealed by patient-derived human-induced pluripotent stem cells

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    Chen, C.; Jiang, P; Xue, H.; Peterson, SE; Tran, HT; McCann, AE; Parast, MM; Li, S.; Pleasure, DE; Laurent, LC; Loring, JF; Liu, Y; Deng, W.

    2014-01-01

    Down's syndrome (DS), caused by trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. Here we use induced pluripotent stem cells (iPSCs) derived from DS patients to identify a role for astrocytes in DS pathogenesis. DS astroglia exhibit higher levels of reactive oxygen species and lower levels of synaptogenic molecules. Astrocyte-conditioned medium collected from DS astroglia causes toxicity to neurons, and fails to promote neuronal ion channel maturatio...

  13. Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction.

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    Morin, Timothy R; Ghassem-Zadeh, Sean A; Lee, Juliet

    2014-08-15

    Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. "Recoil" retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In "pull" type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. "Continuous" type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, "release" type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Cross-Platform Assessment of Genomic Imbalance Confirms the Clinical Relevance of Genomic Complexity and Reveals Loci with Potential Pathogenic Roles in Diffuse Large B-Cell Lymphoma

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    Dias, Lizalynn M.; Thodima, Venkata; Friedman, Julia; Ma, Charles; Guttapalli, Asha; Mendiratta, Geetu; Siddiqi, Imran N.; Syrbu, Sergei; Chaganti, R. S. K.; Houldsworth, Jane

    2016-01-01

    Genomic copy number alterations (CNAs) in diffuse large B-cell lymphoma (DLBCL) have roles in disease pathogenesis but overall clinical relevance remains unclear. Herein, an unbiased algorithm was uniformly applied across three genome profiling datasets comprising 392 newly-diagnosed DLBCL specimens that defined 32 overlapping CNAs, involving 36 minimal common regions (MCRs). Scoring criteria were established for 50 aberrations within the MCRs while considering peak gains/losses. Application of these criteria to independent datasets revealed novel candidate genes with coordinated expression, such as CNOT2, potentially with pathogenic roles. No one single aberration significantly associated with patient outcome across datasets, but genomic complexity, defined by imbalance in more than one MCR, significantly portended adverse outcome in two of three independent datasets. Thus, the standardized scoring of CNAs currently developed can be uniformly applied across platforms, affording robust validation of genomic imbalance and complexity in DLBCL and overall clinical utility as biomarkers of patient outcome. PMID:26294112

  19. FACS Purification and Transcriptome Analysis of Drosophila Neural Stem Cells Reveals a Role for Klumpfuss in Self-Renewal

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    Christian Berger

    2012-08-01

    Full Text Available Drosophila neuroblasts (NBs have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well.

  20. A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells

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    Panarella, Angela; Bexiga, Mariana G.; Galea, George; O’ Neill, Elaine D.; Salvati, Anna; Dawson, Kenneth A.; Simpson, Jeremy C.

    2016-01-01

    Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies. PMID:27374232

  1. An siRNA Screen in Pancreatic Beta Cells Reveals a Role for Gpr27 in Insulin Production

    OpenAIRE

    Ku, Gregory M.; Zachary Pappalardo; Chun Chieh Luo; German, Michael S.; McManus, Michael T.

    2012-01-01

    The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human i...

  2. 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

  3. Genes expressed in the Drosophila head reveal a role for fat cells in sex-specific physiology

    OpenAIRE

    Fujii, Shinsuke; Amrein, Hubert

    2002-01-01

    The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Here, we employed serial analysis of gene expression (SAGE) to identify male and female effectors expressed in the head, and report 46 sex-biased genes (>4-fold/P < 0.01). We characterized four novel, male- or female-specific genes and found that all are expressed mainly in the fat cells in the head. Tsx (turn on sex-spe...

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion

    OpenAIRE

    Russell, Mark A.; Morgan, Noel G

    2011-01-01

    Abstract Common polymorphisms within the fat mass and obesity associated (FTO) gene correlate with increased BMI and a rising risk of type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues including the endocrine pancreas, although its function there is unclear. The current work aimed to investigate the role of FTO in pancreatic ?-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected...

  9. Turgor Regulation in Osmotically Stressed Arabidopsis Epidermal Root Cells. Direct Support for the Role of Inorganic Ion Uptake as Revealed by Concurrent Flux and Cell Turgor Measurements1

    Science.gov (United States)

    Shabala, Sergey N.; Lew, Roger R.

    2002-01-01

    Hyperosmotic stress is known to significantly enhance net uptake of inorganic ions into plant cells. Direct evidence for cell turgor recovery via such a mechanism, however, is still lacking. In the present study, we performed concurrent measurements of net ion fluxes (with the noninvasive microelectrode ion flux estimation technique) and cell turgor changes (with the pressure-probe technique) to provide direct evidence that inorganic ion uptake regulates turgor in osmotically stressed Arabidopsis epidermal root cells. Immediately after onset of hyperosmotic stress (100/100 mm mannitol/sorbitol treatment), the cell turgor dropped from 0.65 to about 0.25 MPa. Turgor recovery started within 2 to 10 min after the treatment and was accompanied by a significant (30–80 nmol m−2 s−1) increase in uptake of K+, Cl−, and Na+ by root cells. In most cells, almost complete (>90% of initial values) recovery of the cell turgor was observed within 40 to 50 min after stress onset. In another set of experiments, we combined the voltage-clamp and the microelectrode ion flux estimation techniques to show that this process is, in part, mediated by voltage-gated K+ transporters at the cell plasma membrane. The possible physiological significance of these findings is discussed. PMID:12011359

  10. 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.

  11. 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.

  12. 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.

  13. Bone marrow chimeras and c-fms conditional ablation (Mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation.

    Science.gov (United States)

    Chinnery, Holly R; Carlson, Eric C; Sun, Yan; Lin, Michelle; Burnett, Sandra H; Perez, Victor L; McMenamin, Paul G; Pearlman, Eric

    2009-03-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4(-/-) mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)(+)/C57BL/6 or eGFP(+)/TLR4(-/-) mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4(-/-) mice reconstituted with C57BL/6, but not TLR4(-/-) bone marrow cells donor cells were found to cause infiltration of eGFP(+) cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP(+) cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1alpha production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation. PMID:19234168

  14. Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation1

    Science.gov (United States)

    Chinnery, Holly R.; Carlson, Eric C.; Sun, Yan; Lin, Michelle; Burnett, Sandra H.; Perez, Victor L.; McMenamin, Paul G.; Pearlman, Eric

    2012-01-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation. PMID:19234168

  15. 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.

  16. 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.

  17. Analyzing gene expression from whole tissue vs. different cell types reveals the central role of neurons in predicting severity of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Shiri Stempler

    Full Text Available Alterations in gene expression resulting from Alzheimer's disease have received considerable attention in recent years. Although expression has been investigated separately in whole brain tissue, in astrocytes and in neurons, a rigorous comparative study quantifying the relative utility of these sources in predicting the progression of Alzheimer's disease has been lacking. Here we analyze gene expression from neurons, astrocytes and whole tissues across different brain regions, and compare their ability to predict Alzheimer's disease progression by building pertaining classification models based on gene expression sets annotated to different biological processes. Remarkably, we find that predictions based on neuronal gene expression are significantly more accurate than those based on astrocyte or whole tissue expression. The findings explicate the central role of neurons, particularly as compared to glial cells, in the pathogenesis of Alzheimer's disease, and emphasize the importance of measuring gene expression in the most relevant (pathogenically 'proximal' single cell types.

  18. 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%.

  19. 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

  20. 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.

  1. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Protein-Protein Interaction Inhibitor Reveals a Non-catalytic Role for GAPDH Oligomerization in Cell Death.

    Science.gov (United States)

    Qvit, Nir; Joshi, Amit U; Cunningham, Anna D; Ferreira, Julio C B; Mochly-Rosen, Daria

    2016-06-24

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme, has a non-catalytic (thus a non-canonical) role in inducing mitochondrial elimination under oxidative stress. We recently demonstrated that phosphorylation of GAPDH by δ protein kinase C (δPKC) inhibits this GAPDH-dependent mitochondrial elimination. δPKC phosphorylation of GAPDH correlates with increased cell injury following oxidative stress, suggesting that inhibiting GAPDH phosphorylation should decrease cell injury. Using rational design, we identified pseudo-GAPDH (ψGAPDH) peptide, an inhibitor of δPKC-mediated GAPDH phosphorylation that does not inhibit the phosphorylation of other δPKC substrates. Unexpectedly, ψGAPDH decreased mitochondrial elimination and increased cardiac damage in an animal model of heart attack. Either treatment with ψGAPDH or direct phosphorylation of GAPDH by δPKC decreased GAPDH tetramerization, which corresponded to reduced GAPDH glycolytic activity in vitro and ex vivo Taken together, our study identified the potential mechanism by which oxidative stress inhibits the protective GAPDH-mediated elimination of damaged mitochondria. Our study also identified a pharmacological tool, ψGAPDH peptide, with interesting properties. ψGAPDH peptide is an inhibitor of the interaction between δPKC and GAPDH and of the resulting phosphorylation of GAPDH by δPKC. ψGAPDH peptide is also an inhibitor of GAPDH oligomerization and thus an inhibitor of GAPDH glycolytic activity. Finally, we found that ψGAPDH peptide is an inhibitor of the elimination of damaged mitochondria. We discuss how this unique property of increasing cell damage following oxidative stress suggests a potential use for ψGAPDH peptide-based therapy. PMID:27129213

  2. 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

  3. 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.

  4. Reassessment of an Arabidopsis cell wall invertase inhibitor AtCIF1 reveals its role in seed germination and early seedling growth.

    Science.gov (United States)

    Su, Tao; Wolf, Sebastian; Han, Mei; Zhao, Hongbo; Wei, Hongbin; Greiner, Steffen; Rausch, Thomas

    2016-01-01

    In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) are recognized as essential players in sugar metabolism and sugar signaling, thereby affecting source-sink interactions, plant development and responses to environmental cues. CWI and VI expression levels are transcriptionally controlled; however, both enzymes are also subject to posttranslational control by invertase inhibitor proteins. The physiological significances of inhibitor proteins during seed germination and early seedling development are not yet fully understood. Here, we demonstrate that the inhibitor isoform AtCIF1 impacted on seed germination and early seedling growth in Arabidopsis. The primary target of AtCIF1 was shown to be localized to the apoplast after expressing an AtCIF1 YFP-fusion construct in tobacco epidermis and transgenic Arabidopsis root. The analysis of expression patterns showed that AtCWI1 was co-expressed spatiotemporally with AtCIF1 within the early germinating seeds. Seed germination was observed to be accelerated independently of exogenous abscisic acid (ABA) in the AtCIF1 loss-of-function mutant cif1-1. This effect coincided with a drastic increase of CWI activity in cif1-1 mutant seeds by 24 h after the onset of germination, both in vitro and in planta. Accordingly, quantification of sugar content showed that hexose levels were significantly boosted in germinating seeds of the cif1-1 mutant. Further investigation of AtCIF1 overexpressors in Arabidopsis revealed a markedly suppressed CWI activity as well as delayed seed germination. Thus, we conclude that the posttranslational modulation of CWI activity by AtCIF1 helps to orchestrate seed germination and early seedling growth via fine-tuning sucrose hydrolysis and, possibly, sugar signaling. PMID:26546341

  5. 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...... cytometry screen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC) function, and JAM-A expression was reduced in normal...

  6. 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.

  7. Three-dimensional culture of sebaceous gland cells revealing the role of prostaglandin E2-induced activation of canonical Wnt signaling

    International Nuclear Information System (INIS)

    Highlights: •Three-dimensional culture generates “semi-vivo” sebaceous glands. •Xenograft model failed to mimic the biology of sebaceous glands in vivo. •Proinflammatory cytokine PGE2 enhances Wnt signal activity in the organoids. •PGE2 influences on the mitochondrial and lipid metabolism in the organoids. •Considering 3R agenda, “semi-vivo” sebaceous glands are useful for research. -- Abstract: Background: Prostaglandin E2 (PGE2) 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 PGE2 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 PGE2 were larger and adopted a more complex organization compared with control organoids. PGE2 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 PGE2 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 PGE2-induced activation of canonical Wnt signaling pathway in regulation of the morphology,proliferation, and function of “semi-vivo” sebaceous glands

  8. Optogenetic Manipulation of Activity and Temporally Controlled Cell-Specific Ablation Reveal a Role for MCH Neurons in Sleep/Wake Regulation

    OpenAIRE

    Tsunematsu, Tomomi; Ueno, Takafumi; Tabuchi, Sawako; Inutsuka, Ayumu; Tanaka, Kenji F.; HASUWA, Hidetoshi; Kilduff, Thomas S.; Terao, Akira; Yamanaka, Akihiro

    2014-01-01

    Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generate...

  9. 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.

  10. 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

  11. Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement.

    Science.gov (United States)

    Katano, Mana; Takahashi, Kazuki; Hirano, Tomonari; Kazama, Yusuke; Abe, Tomoko; Tsukaya, Hirokazu; Ferjani, Ali

    2016-01-01

    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 (12)C(6+) 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 seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post

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

    Directory of Open Access Journals (Sweden)

    Justin D. Lathia

    2014-01-01

    Full Text Available 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 cytometry screen on patient-derived glioblastoma (GBM cells and identified junctional adhesion molecule A (JAM-A as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC function, and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromised the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate that GBM-targeting strategies can be identified through screening adhesion receptors and JAM-A represents a mechanism for niche-driven CSC maintenance.

  13. 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.

  14. 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

  15. 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

    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...

  16. Investigation of triterpene synthesis and regulation in oats reveals a role for β-amyrin in determining root epidermal cell patterning.

    Science.gov (United States)

    Kemen, Ariane C; Honkanen, Suvi; Melton, Rachel E; Findlay, Kim C; Mugford, Sam T; Hayashi, Keiko; Haralampidis, Kosmas; Rosser, Susan J; Osbourn, Anne

    2014-06-10

    Sterols have important functions in membranes and signaling. Plant sterols are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to cycloartenol. Plants also convert 2,3-oxidosqualene to other sterol-like cyclization products, including the simple triterpene β-amyrin. The function of β-amyrin per se is unknown, but this molecule can serve as an intermediate in the synthesis of more complex triterpene glycosides associated with plant defense. β-Amyrin is present at low levels in the roots of diploid oat (Avena strigosa). Oat roots also synthesize the β-amyrin-derived triterpene glycoside avenacin A-1, which provides protection against soil-borne diseases. The genes for the early steps in avenacin A-1 synthesis [saponin-deficient 1 and 2 (Sad1 and Sad2)] have been recruited from the sterol pathway by gene duplication and neofunctionalization. Here we show that Sad1 and Sad2 are regulated by an ancient root developmental process that is conserved across diverse species. Sad1 promoter activity is dependent on an L1 box motif, implicating sterol/lipid-binding class IV homeodomain leucine zipper transcription factors as potential regulators. The metabolism of β-amyrin is blocked in sad2 mutants, which therefore accumulate abnormally high levels of this triterpene. The accumulation of elevated levels of β-amyrin in these mutants triggers a "superhairy" root phenotype. Importantly, this effect is manifested very early in the establishment of the root epidermis, causing a greater proportion of epidermal cells to be specified as root hair cells rather than nonhair cells. Together these findings suggest that simple triterpenes may have widespread and as yet largely unrecognized functions in plant growth and development.

  17. 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.

  18. 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

    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...... 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...

  19. 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

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

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

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

  1. 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...

  2. 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.

  3. NMR metabolomic profiling reveals new roles of SUMOylation in DNA damage response.

    Science.gov (United States)

    Cano, Kristin E; Li, Yi-Jia; Chen, Yuan

    2010-10-01

    Post-translational modifications by the Small Ubiquitin-like Modifier (SUMO) family of proteins have been established as critical events in the cellular response to a wide range of DNA damaging reagents and radiation; however, the detailed mechanism of SUMOylation in DNA damage response is not well understood. In this study, we used a nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach to examine the effect of an inhibitor of SUMO-mediated protein-protein interactions on MCF7 breast cancer cell response to radiation. Metabolomics is sensitive to changes in cellular functions and thus provides complementary information to other biological studies. The peptide inhibitor (SUMO interaction motif mimic, SIM) and a control peptide were stably expressed in MCF-7 cell line. Metabolite profiles of the cell lines before and after radiation were analyzed using solution NMR methods. Various statistical methods were used to isolate significant changes. Differences in the amounts of glutamine, aspartate, malate, alanine, glutamate and NADH between the SIM-expressing and control cells suggest a role for SUMOylation in regulating mitochondrial function. This is also further verified following the metabolism of (13)C-labeled glutamine. The inability of the cells expressing the SIM peptide to increase production of the antioxidants carnosine and glutathione after radiation damage suggests an important role of SUMOylation in regulating the levels of antioxidants that protect cells from free radicals and reactive oxygen species generated by radiation. This study reveals previously unknown roles of SUMOylation in DNA damage response. PMID:20695451

  4. 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

  5. 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.

  6. 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

  7. Adhesion in the stem cell niche: biological roles and regulation

    OpenAIRE

    Chen, Shuyi; Lewallen, Michelle; Xie, Ting

    2013-01-01

    Stem cell self-renewal is tightly controlled by the concerted action of stem cell-intrinsic factors and signals within the niche. Niche signals often function within a short range, allowing cells in the niche to self-renew while their daughters outside the niche differentiate. Thus, in order for stem cells to continuously self-renew, they are often anchored in the niche via adhesion molecules. In addition to niche anchoring, however, recent studies have revealed other important roles for adhe...

  8. 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.

  9. 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. PMID:23733601

  10. Physiological roles revealed by ghrelin and ghrelin receptor deficient mice

    Science.gov (United States)

    Ghrelin is a hormone made in the stomach and known primarily for its growth hormone releasing and orexigenic properties. Nevertheless, ghrelin through its receptor, the GHS-R1a, has been shown to exert many roles including regulation of glucose homeostasis, memory & learning, food addiction and neur...

  11. 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.

  12. 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.

  13. 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.

  14. 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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-02-01

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

  17. 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.

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

    Science.gov (United States)

    Fang, Chao; Li, Lei; Li, Jianmin

    2016-01-01

    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 Ppp2ca(flox/flox) transgenic mice, and conditionally knocked out Ppp2ca in the epidermis driven by Krt14-Cre. Our study showed that Ppp2ca(flox/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, Ppp2ca(flox/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 Ppp2ca(flox/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. PMID:27213341

  19. Role of cancer stem cells in hepatocarcinogenesis

    OpenAIRE

    Wang, Bo; Jacob, Samson T.

    2011-01-01

    There has been considerable interest in cancer stem cells (CSCs) among cancer biologists and clinicians, most likely because of their role in the heterogeneity of cancer and their potential application in cancer therapeutics. Recent studies suggest that CSCs play a key role in liver carcinogenesis. A small subpopulation of cancer cells with CSC properties has been identified and characterized from hepatocellular carcinoma (HCC) cell lines, animal models and human primary HCCs. Considering the...

  20. Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity.

    NARCIS (Netherlands)

    Forcet, C.; Etienne-Manneville, S.; Gaude, H.; Fournier, L.; Debilly, S.; Salmi, M.; Baas, A.; Olschwang, S.; Clevers, J.C.; Billaud, M.

    2005-01-01

    Germline mutations of the LKB1 gene are responsible for the cancer-prone Peutz-Jeghers syndrome (PJS). LKB1 encodes a serine-threonine kinase that acts as a regulator of cell cycle, metabolism and cell polarity. The majority of PJS missense mutations abolish LKB1 enzymatic activity and thereby impai

  1. 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

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

    Science.gov (United States)

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

    2016-07-01

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

  3. Multisystem Langerhans Cell Histiocytosis in Adults Revealed by Skin Lesions.

    Science.gov (United States)

    Atarguine, Hanane; Hocar, Ouafa; Oussmane, Samia; Mouafik, Sara Batoul; Hamdaoui, Abderrachid; Hafiane, Hanan; Belbaraka, Rhizlane; Akhdari, Nadia; Amal, Said

    2016-01-01

    A 37-year-old woman with no remarkable medical or family history presented with papules and vesicles on an erythematous background involving the neck, sacrum, and folds (postauricular, axillary, inguinal, and under the breasts) (Figure 1). During the previous year, she was treated with local and systemic antifungals without improvement. Her history included a secondary amenorrhea, polydipsia, and polyuria (6 L/d) that started 2 years prior. Physical examination revealed chronic bilateral purulent otorrhea with thick eardrums. Histologic examination of skin biopsy revealed a highly suggestive appearance of multisystem Langerhans cell histiocytosis (LCH) with immunohistochemistry (anti-PS100 and anti-CD1a), which were positive (Figure 2A and 2B). Pituitary magnetic resonance imaging showed a thickening of the pituitary stalk in relation to a location histiocytic (Figure 3). Bone gaps were objectified on two radiographic tibial diaphyseal. Results from computed tomography (CT) scan showed a magma coelio mesenteric, axillary, and inguinal lymph nodes. PMID:27319965

  4. 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.

  5. Automatic morphological subtyping reveals new roles of caspases in mitochondrial dynamics.

    Directory of Open Access Journals (Sweden)

    Jyh-Ying Peng

    2011-10-01

    Full Text Available Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.

  6. Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.

    Science.gov (United States)

    Zeaiter, Zaher; Cohen, David; Müsch, Anne; Bagnoli, Fabio; Covacci, Antonello; Stein, Markus

    2008-03-01

    Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation. PMID:18005242

  7. 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

  8. Lmo mutants reveal a novel role for circadian pacemaker neurons in cocaine-induced behaviors.

    Directory of Open Access Journals (Sweden)

    Linus T-Y Tsai

    2004-12-01

    Full Text Available Drosophila has been developed recently as a model system to investigate the molecular and neural mechanisms underlying responses to drugs of abuse. Genetic screens for mutants with altered drug-induced behaviors thus provide an unbiased approach to define novel molecules involved in the process. We identified mutations in the Drosophila LIM-only (LMO gene, encoding a regulator of LIM-homeodomain proteins, in a genetic screen for mutants with altered cocaine sensitivity. Reduced Lmo function increases behavioral responses to cocaine, while Lmo overexpression causes the opposite effect, reduced cocaine responsiveness. Expression of Lmo in the principal Drosophila circadian pacemaker cells, the PDF-expressing ventral lateral neurons (LN(vs, is sufficient to confer normal cocaine sensitivity. Consistent with a role for Lmo in LN(vfunction,Lmomutants also show defects in circadian rhythms of behavior. However, the role for LN(vs in modulating cocaine responses is separable from their role as pacemaker neurons: ablation or functional silencing of the LN(vs reduces cocaine sensitivity, while loss of the principal circadian neurotransmitter PDF has no effect. Together, these results reveal a novel role for Lmo in modulating acute cocaine sensitivity and circadian locomotor rhythmicity, and add to growing evidence that these behaviors are regulated by shared molecular mechanisms. The finding that the degree of cocaine responsiveness is controlled by the Drosophila pacemaker neurons provides a neuroanatomical basis for this overlap. We propose that Lmo controls the responsiveness of LN(vs to cocaine, which in turn regulate the flies' behavioral sensitivity to the drug.

  9. DEFECTIVE ENGRAFTMENT OF C3aR-/- HEMATOPOIETIC STEM PROGENITOR CELLS REVEALS A NOVEL ROLE OF THE C3a-C3aR AXIS IN BONE MARROW HOMING

    OpenAIRE

    Wysoczynski, Marcin; Reca, Ryan; Lee, HakMo; Wu, Wan; Ratajczak, Janina; Mariusz Z Ratajczak

    2009-01-01

    We reported that complement (C) becomes activated and cleaved in bone marrow (BM) during preconditioning for hematopoietic transplantation and the third C component (C3) cleavage fragments C3a and desArgC3a increase responsiveness of hematopoietic stem/progenitor cells (HSPCs) to stromal derived factor-1 (SDF-1). We also demonstrated that this homing promoting effect is not C3a receptor (C3aR) dependent. Herein, we report our new observation that transplantation of C3aR-/- HSPCs into lethally...

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

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

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

  11. 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.

  12. 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.

  13. Switching roles: the functional plasticity of adult tissue stem cells.

    Science.gov (United States)

    Wabik, Agnieszka; Jones, Philip H

    2015-05-01

    Adult organisms have to adapt to survive, and the same is true for their tissues. Rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges. Recent work reveals how stem cell (SC) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration. This plasticity extends to differentiating cells, which are capable of reverting to SCs after injury. The concept of the niche, the micro-environment that sustains and regulates stem cells, is broadening, with a new appreciation of the role of physical factors and hormonal signals. Here, we review different functions of SCs, the cellular mechanisms that underlie them and the signals that bias the fate of SCs as they switch between roles. PMID:25812989

  14. 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.

  15. 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

    . 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......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...... 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...

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-05-21

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

  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. 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.

  6. 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.

  7. 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.

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

    OpenAIRE

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

    2012-01-01

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

  9. 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

  10. 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.

  11. Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation.

    Science.gov (United States)

    Babazadeh, Roja; Furukawa, Takako; Hohmann, Stefan; Furukawa, Kentaro

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data. PMID:24732094

  12. 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

  13. 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.

  14. 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.

  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. 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.

  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...

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

    Directory of Open Access Journals (Sweden)

    Lulu Cao

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

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Simpson David A

    2010-05-01

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

  1. 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.

  2. Transcriptome sequencing reveals the roles of transcription factors in modulating genotype by nitrogen interaction in maize

    OpenAIRE

    Chen, Qiuyue; Liu, Zhipeng; Wang, Baobao; Wang, Xufeng; Lai, Jinsheng; Tian, Feng

    2015-01-01

    Key message Global transcriptome analysis in maize revealed differential nitrogen response between genotypes and implicate a crucial role of transcription factors in driving genotype by nitrogen interactions at gene expression level. Abstract Developing nitrogen-efficient cultivars are essential for sustainable and productive agriculture. Nitrogen use efficiency of plants is highly dependent on the interaction of environmental and genetic variation and results in adaptive phenotypes. This stu...

  3. Live cell imaging reveals at novel view of DNA

    International Nuclear Information System (INIS)

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

  4. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

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    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

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

    OpenAIRE

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

    2016-01-01

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

  6. Geometric determinants of directional cell motility revealed using microcontact printing.

    Science.gov (United States)

    Brock, Amy; Chang, Eric; Ho, Chia-Chi; LeDuc, Philip; Jiang, Xingyu; Whitesides, George M; Ingber, Donald E

    2003-03-01

    Mammalian cells redirect their movement in response to changes in the physical properties of their extracellular matrix (ECM) adhesive scaffolds, including changes in available substrate area, shape, or flexibility. Yet, little is known about the cell's ability to discriminate between different types of spatial signals. Here we utilize a soft-lithography-based, microcontact printing technology in combination with automated computerized image analysis to explore the relationship between ECM geometry and directional motility. When fibroblast cells were cultured on fibronectin-coated adhesive islands with the same area (900 micrometers2) but different geometric forms (square, triangle, pentagon, hexagon, trapezoid, various parallelograms) and aspect ratios, cells preferentially extended new lamellipodia from their corners. In addition, by imposing these simple geometric constraints through ECM, cells were directed to deposit new fibronectin fibrils in these same corner regions. These data indicate that mammalian cells can sense edges within ECM patterns that exhibit a wide range of angularity and that they use these spatial cues to guide where they will deposit ECM and extend new motile processes during the process of directional migration. PMID:14674434

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Role of polyphenols in cell death control.

    Science.gov (United States)

    Giovannini, Claudio; Masella, Roberta

    2012-05-01

    Dietary consumption of fruit, vegetables, fish, and olive oil has been demonstrated to exert beneficial effects on human health. This finding may be due to the high content of antioxidant compounds including polyphenols. Current evidence strongly supports a contribution of polyphenols to the prevention of several chronic degenerative diseases such as cancer, atherosclerosis and cardiovascular diseases, central nervous system disorders, as well as aging. Apoptosis is a genetically controlled and evolutionarily conserved form of cell death of critical importance for the maintenance of tissue homeostasis in the adult organism. The malfunction of the death machinery may play a primary role in various pathologic processes, leading to proliferative or degenerative diseases. Polyphenols can interact with specific steps and/or proteins regulating the apoptotic process in different ways depending on their concentration, the cell system, the type or stage of the pathological process. Because of their ability to modulate cell death, polyphenols have been proposed as chemopreventive and therapeutic agents. This paper reviews and discusses the last 3-year findings related to the principal molecular mechanisms involved in the control of the balance between apoptosis and cell proliferation exerted by polyphenols. PMID:22584012

  9. 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.

  10. The enigmatic role of mast cells in dominant tolerance

    OpenAIRE

    de Vries, Victor C.; Pino-Lagos, Karina; Elgueta, Raul; Noelle, Randolph J.

    2009-01-01

    PURPOSE OF REVIEW: The role of regulatory T cells (Treg) in peripheral tolerance has been studied extensively in transplantation research. Recently, mast cells have been shown to play an indispensable role in allograft tolerance. The purpose of this review is to inform the reader on the current standings of the role of mast cells in dominant tolerance with an emphasis on the interaction of mast cells with Treg.RECENT FINDINGS: Mast cells are required to sustain peripheral tolerance via Treg. ...

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

    OpenAIRE

    Reboul, Rebecca; Tenhaken, Raimund

    2012-01-01

    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 pre...

  12. Functional role of regulatory T cells in B cell lymphoma and related mechanisms.

    Science.gov (United States)

    Wu, Wei; Wan, Jun; Xia, Ruixiang; Huang, Zhenqi; Ni, Jing; Yang, Mingzhen

    2015-01-01

    B cell lymphoma (BCL) has a higher degree of malignancy and complicated pathogenic mechanism. Regulatory T cells (Treg cells) are known to exert certain immune suppression functions, in addition to immune mediating effects. Recent studies have revealed the role of Treg cells in pathogenesis and progression of multiple malignant tumors. This study therefore investigated the functional role and related mechanism of Treg cells in BCL. A cohort of thirty patients who were diagnosed with BCL in our hospital between January 2013 and December 2014. Another thirty healthy individuals were recruited. Peripheral blood mononuclear cells (PBMCs) were separated and analyzed for the ratio of CD4+/CD25+ Treg cells. The mRNA expression levels of Foxp3, transforming growth factor (TGF)-β1 and interleukin (IL)-10 genes were quantified by real-time PCR, while their serum levels were determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile all laboratory indexes for patients were monitored during the complete remission (CR) stage. BCL patients significantly elevated ratio of CD4+/CD25+ Treg cells, which were decreased at CR stage. mRNA levels of Foxp3, TGF-β1 and IL-10, in addition to protein levels of TGF-β1 and IL-10 were potentiated in lymphoma patients but decreased in CR patients (Pregulating cytokines, thereby facilitating the pathogenesis and progression of lymphoma. PMID:26464657

  13. 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.

  14. 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.

  15. NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

    Science.gov (United States)

    Proto, J D; Tang, Y; Lu, A; Chen, W C W; Stahl, E; Poddar, M; Beckman, S A; Robbins, P D; Nidernhofer, L J; Imbrogno, K; Hannigan, T; Mars, W M; Wang, B; Huard, J

    2015-01-01

    The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD. PMID:25906153

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  18. 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

  19. 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.

  20. Network Analysis of a Comprehensive Knowledge Repository Reveals a Dual Role for Ceramide in Alzheimer's Disease.

    Science.gov (United States)

    Mizuno, Satoshi; Ogishima, Soichi; Kitatani, Kazuyuki; Kikuchi, Masataka; Tanaka, Hiroshi; Yaegashi, Nobuo; Nakaya, Jun

    2016-01-01

    Alzheimer's disease (AD) is the most common cause of senile dementia. Many inflammatory factors such as amyloid-β and pro-inflammatory cytokines are known to contribute to the inflammatory response in the AD brain. Sphingolipids are widely known to have roles in the pathogenesis of inflammatory diseases, where the precise roles for sphingolipids in inflammation-associated pathogenesis of AD are not well understood. Here we performed a network analysis to clarify the importance of sphingolipids and to model relationships among inflammatory factors and sphingolipids in AD. In this study, we have updated sphingolipid signaling and metabolic cascades in a map of AD signaling networks that we named "AlzPathway," a comprehensive knowledge repository of signaling pathways in AD. Our network analysis of the updated AlzPathway indicates that the pathways related to ceramide are one of the primary pathways and that ceramide is one of the important players in the pathogenesis of AD. The results of our analysis suggest the following two prospects about inflammation in AD: (1) ceramide could play important roles in both inflammatory and anti-inflammatory pathways of AD, and (2) several factors such as Sphingomyelinase and Siglec-11 may be associated with ceramide related inflammation and anti-inflammation pathways in AD. In this study, network analysis of comprehensive knowledge repository reveals a dual role for ceramide in AD. This result provides a clue to clarify sphingolipids related inflammatory and anti-inflammatory pathways in AD. PMID:26849355

  1. Network Analysis of a Comprehensive Knowledge Repository Reveals a Dual Role for Ceramide in Alzheimer's Disease.

    Directory of Open Access Journals (Sweden)

    Satoshi Mizuno

    Full Text Available Alzheimer's disease (AD is the most common cause of senile dementia. Many inflammatory factors such as amyloid-β and pro-inflammatory cytokines are known to contribute to the inflammatory response in the AD brain. Sphingolipids are widely known to have roles in the pathogenesis of inflammatory diseases, where the precise roles for sphingolipids in inflammation-associated pathogenesis of AD are not well understood. Here we performed a network analysis to clarify the importance of sphingolipids and to model relationships among inflammatory factors and sphingolipids in AD. In this study, we have updated sphingolipid signaling and metabolic cascades in a map of AD signaling networks that we named "AlzPathway," a comprehensive knowledge repository of signaling pathways in AD. Our network analysis of the updated AlzPathway indicates that the pathways related to ceramide are one of the primary pathways and that ceramide is one of the important players in the pathogenesis of AD. The results of our analysis suggest the following two prospects about inflammation in AD: (1 ceramide could play important roles in both inflammatory and anti-inflammatory pathways of AD, and (2 several factors such as Sphingomyelinase and Siglec-11 may be associated with ceramide related inflammation and anti-inflammation pathways in AD. In this study, network analysis of comprehensive knowledge repository reveals a dual role for ceramide in AD. This result provides a clue to clarify sphingolipids related inflammatory and anti-inflammatory pathways in AD.

  2. 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.

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

    International Nuclear Information System (INIS)

    Highlights: ► We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. ► Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. ► Autophagy was required for normal terminal differentiation of preputial gland cells. ► 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.

  4. 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.

  5. 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 retinal...

  6. 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

  7. Physical role for the nucleus in cell migration.

    Science.gov (United States)

    Fruleux, Antoine; Hawkins, Rhoda J

    2016-09-14

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration. PMID:27406341

  8. Physical role for the nucleus in cell migration

    Science.gov (United States)

    Fruleux, Antoine; Hawkins, Rhoda J.

    2016-09-01

    Cell migration is important for the function of many eukaryotic cells. Recently the nucleus has been shown to play an important role in cell motility. After giving an overview of cell motility mechanisms we review what is currently known about the mechanical properties of the nucleus and the connections between it and the cytoskeleton. We also discuss connections to the extracellular matrix and mechanotransduction. We identify key physical roles of the nucleus in cell migration.

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

    OpenAIRE

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

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Roland Keller

    2016-01-01

    Full Text Available 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.

  11. 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.

  12. NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair

    OpenAIRE

    Proto, J D; Tang, Y.; Lu, A.; Chen, W C W; Stahl, E; Poddar, M.; Beckman, S A; Robbins, P. D.; Nidernhofer, L J; Imbrogno, K; Hannigan, T; Mars, W M; Wang, B; Huard, J

    2015-01-01

    The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found tha...

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  15. Role of Calmodulin in Cell Proliferation

    Science.gov (United States)

    Chafouleas, J.

    1983-01-01

    Calmodulin levels were found to increase as cells enter plateau. The data suggest that the cells are exiting the cell cycle late in the G sub 1 phase, or that the calmodulin levels in plateau cells are uncoupled to progression into S phase in plateau cells. Upon release, calmodulin levels rapidly decrease. Following this decrease, there is a increase prior to S phase.

  16. 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.

  17. 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.

  18. Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis

    Directory of Open Access Journals (Sweden)

    Aigner Ludwig

    2008-05-01

    suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of IkappaBalpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation. Conclusion We identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

  19. 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.

  20. 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.

  1. 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.

  2. Role of Vibrational Spectroscopy in Stem Cell Research

    OpenAIRE

    AKSOY, Ceren; Severcan, Feride

    2012-01-01

    Recent researches have mainly displayed the significant role of stem cells in tissue renewal and homeostasis with their unique capacity to develop different cell types. These findings have clarified the importance of stem cells to improve the effectiveness of any cell therapy for regenerative medicine. Identification of purity and differentiation stages of stem cells are the greatest challenges of stem cell biology and regenerative medicine. The existing methods to carefully monitor and chara...

  3. 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. PMID:12506008

  4. 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.

  5. STEM CELLS: Differentiated cells in a back-up role

    OpenAIRE

    Desai, Tushar J.; Krasnow, Mark A.

    2013-01-01

    Two independent studies show that, if push comes to shove, differentiated cells of the stomach and lung can act as adult stem cells generating various cell types of the tissue, including a pool of stem cells.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. The role of mast cells in functional GI disorders.

    Science.gov (United States)

    Wouters, Mira M; Vicario, Maria; Santos, Javier

    2016-01-01

    Functional gastrointestinal disorders (FGIDs) are characterized by chronic complaints arising from disorganized brain-gut interactions leading to dysmotility and hypersensitivity. The two most prevalent FGIDs, affecting up to 16-26% of worldwide population, are functional dyspepsia and irritable bowel syndrome. Their etiopathogenic mechanisms remain unclear, however, recent observations reveal low-grade mucosal inflammation and immune activation, in association with impaired epithelial barrier function and aberrant neuronal sensitivity. These findings come to challenge the traditional view of FGIDs as pure functional disorders, and relate the origin to a tangible organic substrate. The mucosal inflammatory infiltrate is dominated by mast cells, eosinophils and intraepithelial lymphocytes in the intestine of FGIDs. It is well established that mast cell activation can generate epithelial and neuro-muscular dysfunction and promote visceral hypersensitivity and altered motility patterns in FGIDs, postoperative ileus, food allergy and inflammatory bowel disease. This review will discuss the role of mucosal mast cells in the gastrointestinal tract with a specific focus on recent advances in disease mechanisms and clinical management in irritable bowel syndrome and functional dyspepsia. PMID:26194403

  8. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    Directory of Open Access Journals (Sweden)

    Jyuhn-Huarng Juang

    2015-02-01

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

  9. Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation

    Directory of Open Access Journals (Sweden)

    Kingsley David M

    2008-04-01

    Full Text Available Abstract Background Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear. Results Here, we identify a novel germline mutation at the mouse Bone morphogenetic protein 5 (Bmp5 locus. Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5. Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo. Biomechanical studies of osteoblasts from these anatomic sites show that the mutation inhibits the proper response of bone cells to mechanical stimulation. Conclusion The results from these genetic, biochemical, and biomechanical studies suggest that BMPs are required not only for skeletal patterning during embryonic development, but also for bone response and remodeling to mechanical stimulation at specific anatomic sites in the skeleton.

  10. 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.

  11. A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division.

    Directory of Open Access Journals (Sweden)

    Scott A Hoose

    Full Text Available Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.

  12. 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.

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

    Science.gov (United States)

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

    2016-07-26

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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Selective abrogation of the uPA-uPAR interaction in vivo reveals a novel role in suppression of fibrin-associated inflammation

    DEFF Research Database (Denmark)

    Connolly, Brian M; Choi, Eun Young; Gårdsvoll, Henrik;

    2010-01-01

    overall structure of the domain. Analysis of Plau(GFDhu/GFDhu) mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing inflammation secondary to fibrin deposition. In contrast, leukocyte recruitment and tissue regeneration were unaffected by the loss of uPA binding to uPAR. This...... study identifies a principal in vivo role of the uPA-uPAR interaction in cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation and provides a valuable model for further exploration of this multifunctional receptor....

  18. 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.

  19. Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection

    DEFF Research Database (Denmark)

    Ørding Kauffmann, Susanne; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2006-01-01

    or their distribution between lymphoid and nonlymphoid organs. Regarding a functional role of VLA-1, we found that intracerebral infection of both VLA-1(-/-) and wild-type (wt) mice resulted in lethal T-cell-mediated meningitis, and quantitative and qualitative analyses of the cellular exudate did not reveal any...

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

    Science.gov (United States)

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

    2016-07-26

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

  1. 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.

  2. The role of dental stem cells in regeneration

    OpenAIRE

    MAXIM, MONICA ANGELA; Soritau, Olga; BACIUT, MIHAELA; BRAN, SIMION; BACIUT, GRIGORE

    2015-01-01

    Mesenchymal stem cells (MSCs) are adult stem cells that have the capacity of rising multiple cell types. A rich source of mesenchymal stem cells is represented by the dental tissues: the periodontal ligament, the dental pulp, the apical papilla, the dental follicle and the deciduous teeth. The aim of this review is to characterize the main dental- derived mesenchymal stem cell population, and to show their important role in tissue regeneration based on their properties : the multi-potency, th...

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Gas7-deficient mouse reveals roles in motor function and muscle fiber composition during aging.

    Directory of Open Access Journals (Sweden)

    Bo-Tsang Huang

    Full Text Available BACKGROUND: Growth arrest-specific gene 7 (Gas7 has previously been shown to be involved in neurite outgrowth in vitro; however, its actual role has yet to be determined. To investigate the physiological function of Gas7 in vivo, here we generated a Gas7-deficient mouse strain with a labile Gas7 mutant protein whose functions are similar to wild-type Gas7. METHODOLOGY/PRINCIPAL FINDINGS: Our data show that aged Gas7-deficient mice have motor activity defects due to decreases in the number of spinal motor neurons and in muscle strength, of which the latter may be caused by changes in muscle fiber composition as shown in the soleus. In cross sections of the soleus of Gas7-deficient mice, gross morphological features and levels of myosin heavy chain I (MHC I and MHC II markers revealed significantly fewer fast fibers. In addition, we found that nerve terminal sprouting, which may be associated with slow and fast muscle fiber composition, was considerably reduced at neuromuscular junctions (NMJ during aging. CONCLUSIONS/SIGNIFICANCE: These findings indicate that Gas7 is involved in motor neuron function associated with muscle strength maintenance.

  8. 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.

  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. PMID:27507938

  10. 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.

  11. 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

  12. Transcriptome Analysis of Pepper (Capsicum annuum) Revealed a Role of 24-Epibrassinolide in Response to Chilling

    Science.gov (United States)

    Li, Jie; Yang, Ping; Kang, Jungen; Gan, Yantai; Yu, Jihua; Calderón-Urrea, Alejandro; Lyu, Jian; Zhang, Guobin; Feng, Zhi; Xie, Jianming

    2016-01-01

    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

  13. Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates.

    Directory of Open Access Journals (Sweden)

    Matthew P Harris

    Full Text Available The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda and ectodysplasin receptor (edar genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100 that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

  14. Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

    Science.gov (United States)

    Harris, Matthew P.; Rohner, Nicolas; Schwarz, Heinz; Perathoner, Simon; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

    2008-01-01

    The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution. PMID:18833299

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

    OpenAIRE

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

    2015-01-01

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

  16. A coculture model mimicking the intestinal mucosa reveals a regulatory role for myofibroblasts in immune-mediated barrier disruption.

    Science.gov (United States)

    Willemsen, L E M; Schreurs, C C H M; Kroes, H; Spillenaar Bilgen, E J; Van Deventer, S J H; Van Tol, E A F

    2002-10-01

    The pathogenesis of Crohn's disease involves a mucosal inflammatory response affecting the barrier function of the gut. Myofibroblasts directly underlining the intestinal epithelium may have a regulatory role in immune-mediated barrier disruption. A coculture system of T84 epithelial and CCD-18Co myofibroblasts was established in order to mimic the in situ spatial interactions between these cell types and to evaluate their role in barrier: integrity. Lamina propria mononuclear cells (LPMC) were introduced in co- and monocultures. Effects of immune cells on barrier integrity was determined by measuring resistance and permeability for macromolecules. Introduction of LPMC in both culture systems caused a time-dependent decrease in barrier integrity. This was found to be less pronounced in cocultures indicating a regulatory role for mesenchymal cells. The effects were also found to depend on the route of LPMC stimulation. Additional analyses suggested that the regulatory role of myofibroblasts in barrier integrity involves production of growth factors. PMID:12395905

  17. 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.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  20. 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

  1. 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...... a reduced nuclear:cytoplasmic ratio, are more transcriptionally active, and contain abundant cellular organelles. This study also revealed cavitation and potential unfolding of the epiblast. As the ICM forms the epiblast, SSEA1 is lost and VIMENTIN is lost and re-expressed. The D6 blastocyst expressed high...

  2. A Pathogenic Role for CD8+ T Cells in a Spontaneous Model of Demyelinating Disease

    DEFF Research Database (Denmark)

    Brisebois, Marcel; Zehntner, Simone P.; Estrada, José;

    2006-01-01

    Transgenic (Tg) mice that overexpress the costimulatory ligand B7.2/CD86 on microglia spontaneously develop a T cell-mediated demyelinating disease. Characterization of the inflammatory infiltrates in the nervous tissue revealed a predominance of CD8+ T cells, suggesting a prominent role of this T...... pathogenesis. Collectively, our data indicate that the spontaneous demyelinating disease in this animal model occurs as a consequence of an inflammatory response initiated through the activation of CNS-specific CD8+ T cells by Tg expression of B7.2 within the target organ. Thus, autoreactive CD8+ T cells can...

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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. PMID:27503417

  8. 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.

  9. 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.

  10. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function.

    Science.gov (United States)

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  11. 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.

  12. 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.

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

    OpenAIRE

    Gumbleton, Matthew; Kerr, William G.

    2013-01-01

    Natural killer (NK) cells are important for 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 both prevent autoimmunity and acquire lytic capacity. Mouse models have shown important roles for inositol phospholipid signaling in lymphocytes. NK cells from mice with deletion in different members of the inositol ph...

  14. 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

  15. Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Georgia Drakakaki; Glenn Hicks; Natasha Raikhel; Wilhelmina van de Ven; Songqin Pan; Yansong Miao; Junqi Wang; Nana F Keinath; Brent Weatherly; Liwen Jiang; Karin Schumacher

    2012-01-01

    The endomembrane system is a complex and dynamic intracellular trafficking network.It is very challenging to track individual vesicles and their cargos in real time; however,affinity purification allows vesicles to be isolated in their natural state so that their constituent proteins can be identified.Pioneering this approach in plants,we isolated the SYP61 trans-Golgi network compartment and carried out a comprehensive proteomic analysis of its contents with only minimal interference from other organelles.The proteome of SYP61 revealed the association of proteins of unknown function that have previously not been ascribed to this compartment.We identified a complete SYP61 SNARE complex,including regulatory proteins and validated the proteome data by showing that several of these proteins associated with SYP61 in planta.We further identified the SYP121-complex and cellulose synthases,suggesting that SYP61 plays a role in the exocytic trafficking and the transport of cell wall components to the plasma membrane.The presence of proteins of unknown function in the SYP61 proteome including ECHIDNA offers the opportunity to identify novel trafficking components and cargos.The affinity purification of plant vesicles in their natural state provides a basis for further analysis and dissection of complex endomembrane networks.The approach is widely applicable and can afford the study of several vesicle populations in plants,which can be compared with the SYP61 vesicle proteome.

  16. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

    Science.gov (United States)

    Lewis, Wesley R; Malarkey, Erik B; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C; Porath, Jonathan D; Birket, Susan E; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R; Leigh, Margaret W; Zariwala, Maimoona A; Challa, Anil K; Kesterson, Robert A; Rowe, Steven M; Drummond, Iain A; Parant, John M; Hildebrandt, Friedhelm; Porter, Mary E; Yoder, Bradley K; Berbari, Nicolas F

    2016-07-01

    Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in

  17. 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.

  18. 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.

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Gitit Shahaf

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

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

    Science.gov (United States)

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

    2016-03-01

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

  3. Role of autophagy in the regulation of epithelial cell junctions.

    Science.gov (United States)

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  4. 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.

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

    OpenAIRE

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

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-08

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

  7. 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.

  8. Innate Lymphoid Cells: Roles In Tumour Genesis And Progression

    OpenAIRE

    Jovanovic Ivan; Gajovic Nevena; Radosavljevic Gordana; Pantic Jelena; Pejnovic Nada; Arsenijevic Nebojsa; Lukic Miodrag L.

    2015-01-01

    Innate lymphoid cells (ILCs) represent the most recently identified members of the innate immune system. These cells play important roles in inflammation, tissue remodelling and metabolic disease. ILCs can be subdivided into three major groups according to their cytokine production. The role of ILCs in tumourigenesis and tumour progression is not completely clarified. In this review, we discuss whether and how ILCs are involved in tumour genesis, growth and metastasis.

  9. Innate Lymphoid Cells: Roles In Tumour Genesis And Progression

    Directory of Open Access Journals (Sweden)

    Jovanovic Ivan

    2015-06-01

    Full Text Available Innate lymphoid cells (ILCs represent the most recently identified members of the innate immune system. These cells play important roles in inflammation, tissue remodelling and metabolic disease. ILCs can be subdivided into three major groups according to their cytokine production. The role of ILCs in tumourigenesis and tumour progression is not completely clarified. In this review, we discuss whether and how ILCs are involved in tumour genesis, growth and metastasis.

  10. 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.   

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

    OpenAIRE

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

    2014-01-01

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

  12. Switching roles: the functional plasticity of adult tissue stem cells

    OpenAIRE

    Wabik, A.; Jones, P H

    2015-01-01

    Adult organisms have to adapt to survive, and the same is true for their tissues. Rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges. Recent work reveals how stem cell (SC) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration. This plasticity extends to differentiating cells, which are capable of reverting to SCs after injury. The concept...

  13. Role of Crosstalk between Epithelial and Immune Cells, the Epimmunome, in Allergic Rhinitis Pathogenesis.

    Science.gov (United States)

    Kamekura, Ryuta; Yamashita, Keiji; Jitsukawa, Sumito; Nagaya, Tomonori; Ito, Fumie; Ichimiya, Shingo; Himi, Tetsuo

    2016-01-01

    Recently, the prevalence of allergic rhinitis has been dramatically increasing worldwide. As conventional therapies for allergic rhinitis, such as antihistamines, leukotriene receptor antagonists, nasal sprays and allergen immunotherapy, have limitations, the development of new drugs is required. Recent studies have revealed that epithelial cell-derived cytokines, including thymic stromal lymphopoietin, interleukin (IL)-25 and IL-33, are able to control immune cells, such as dendritic cells and T cells, thereby acting as 'master switches' in allergic disease. In addition, new roles have been identified for follicular helper T cells and regulatory B cells in allergic disease, and they are considered to be promising targets for new therapies. Thus, crosstalk between epithelial and immune cells, the epimmunome, underlies the pathogenesis of allergic rhinitis. Greater understanding of the epimmunome may lead to breakthroughs in the development of new treatments for allergic rhinitis and will help us cure many patients suffering from its severe symptoms in the future. PMID:27116609

  14. Molecular signatures of prostate stem cells reveal novel signaling pathways and provide insights into prostate cancer.

    Directory of Open Access Journals (Sweden)

    Roy Blum

    Full Text Available BACKGROUND: The global gene expression profiles of adult and fetal murine prostate stem cells were determined to define common and unique regulators whose misexpression might play a role in the development of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: A distinctive core of transcriptional regulators common to both fetal and adult primitive prostate cells was identified as well as molecules that are exclusive to each population. Elements common to fetal and adult prostate stem cells include expression profiles of Wnt, Shh and other pathways identified in stem cells of other organs, signatures of the aryl-hydrocarbon receptor, and up-regulation of components of the aldehyde dehydrogenase/retinoic acid receptor axis. There is also a significant lipid metabolism signature, marked by overexpression of lipid metabolizing enzymes and the presence of the binding motif for Srebp1. The fetal stem cell population, characterized by more rapid proliferation and self-renewal, expresses regulators of the cell cycle, such as E2f, Nfy, Tead2 and Ap2, at elevated levels, while adult stem cells show a signature in which TGF-beta has a prominent role. Finally, comparison of the signatures of primitive prostate cells with previously described profiles of human prostate tumors identified stem cell molecules and pathways with deregulated expression in prostate tumors including chromatin modifiers and the oncogene, Erg. CONCLUSIONS/SIGNIFICANCE: Our data indicate that adult prostate stem or progenitor cells may acquire characteristics of self-renewing primitive fetal prostate cells during oncogenesis and suggest that aberrant activation of components of prostate stem cell pathways may contribute to the development of prostate tumors.

  15. Langerhans cells and their role in oral mucosal diseases.

    Science.gov (United States)

    Upadhyay, Juhi; Upadhyay, Ram B; Agrawal, Pankaj; Jaitley, Shweta; Shekhar, Rhitu

    2013-09-01

    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. PMID:24251267

  16. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Science.gov (United States)

    Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  17. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    Directory of Open Access Journals (Sweden)

    Diego V Bohórquez

    Full Text Available The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM. Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

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

    NARCIS (Netherlands)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2015-11-15

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

  20. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    Science.gov (United States)

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  1. 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.

  2. 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

    2016-01-01

    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. PMID:27383047

  3. 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

  4. 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

  5. Role of Fetal Stem Cells in Maternal Tissue Regeneration

    OpenAIRE

    Zhong, Jiang F; Weiner, Leslie P.

    2007-01-01

    Microchimerism refers to the status of harboring cells from another individual at low levels. It is well known that cells traffic bidirectionally between fetus and mother during pregnancy. This situation resembles a naturally occurring long lasting fetal stem cell transplantation. The fetus acts as the donor and the mother acts as the recipient. To study the role of microchimerism in tissue regeneration, we constructed a murine microchimerism model with wild type C57BL/6J female mice carrying...

  6. 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

    2016-01-01

    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. PMID:27009448

  7. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    Science.gov (United States)

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  8. 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.

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

    OpenAIRE

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

    2014-01-01

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

  10. Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.

    Science.gov (United States)

    Rivarola, Valeria; Flamenco, Pilar; Melamud, Luciana; Galizia, Luciano; Ford, Paula; Capurro, Claudia

    2010-08-01

    Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes. PMID:20432437

  11. 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.

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

    Science.gov (United States)

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

    2016-08-25

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

  13. 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

  14. 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

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

    Science.gov (United States)

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

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

  16. 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.

  17. The significant role of mast cells in cancer.

    Science.gov (United States)

    Khazaie, Khashayarsha; Blatner, Nichole R; Khan, Mohammad Wasim; Gounari, Fotini; Gounaris, Elias; Dennis, Kristen; Bonertz, Andreas; Tsai, Fu-Nien; Strouch, Matthew J; Cheon, Eric; Phillips, Joseph D; Beckhove, Philipp; Bentrem, David J

    2011-03-01

    Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient. PMID:21287360

  18. 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.

  19. Targeted cell elimination reveals an auxin-guided biphasic mode of lateral root initiation

    Science.gov (United States)

    Marhavý, Peter; Montesinos, Juan Carlos; Abuzeineh, Anas; Van Damme, Daniel; Vermeer, Joop E.M.; Duclercq, Jerôme; Rakusová, Hana; Nováková, Petra; Friml, Jiři; Geldner, Niko; Benková, Eva

    2016-01-01

    To sustain a lifelong ability to initiate organs, plants retain pools of undifferentiated cells with a preserved proliferation capacity. The root pericycle represents a unique tissue with conditional meristematic activity, and its tight control determines initiation of lateral organs. Here we show that the meristematic activity of the pericycle is constrained by the interaction with the adjacent endodermis. Release of these restraints by elimination of endodermal cells by single-cell ablation triggers the pericycle to re-enter the cell cycle. We found that endodermis removal substitutes for the phytohormone auxin-dependent initiation of the pericycle meristematic activity. However, auxin is indispensable to steer the cell division plane orientation of new organ-defining divisions. We propose a dual, spatiotemporally distinct role for auxin during lateral root initiation. In the endodermis, auxin releases constraints arising from cell-to-cell interactions that compromise the pericycle meristematic activity, whereas, in the pericycle, auxin defines the orientation of the cell division plane to initiate lateral roots. PMID:26883363

  20. 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.

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

    OpenAIRE

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Roberto Plebani

    2012-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Kaoru Miyazaki

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

  4. Subsets of regulatory T cells and their roles in allergy.

    Science.gov (United States)

    Zhang, Huiyun; Kong, Hui; Zeng, Xiaoning; Guo, Lianyi; Sun, Xiaoyun; He, Shaoheng

    2014-01-01

    In recent years, it is recognized that acquired immunity is controlled by regulatory T cell (Treg). Since fundamental pathophysiological changes of allergy are mainly caused by hyperresponsiveness of immune system to allergens that acquires after birth, Tregs likely play key roles in the pathogenesis of allergy, particularly during the sensitization phase. However, accumulated information indicate that there are several distinctive subtypes of Tregs in man, and each of them seems to play different role in controlling immune system, which complicates the involvement of Tregs in allergy. The aim of the present study is to attempt to classify subtypes of Tregs and summarize their roles in allergy. Tregs should include natural Tregs (nTreg) including inducible costimulator (ICOS)(+) Tregs, inducible/adaptive Tregs (iTreg), interleukin (IL)-10-producing type 1 Tregs (Tr1 cells), CD8(+) Tregs and IL-17-producing Tregs. These cells share some common features including expression of Foxp3 (except for Tr1 cells), and secretion of inhibitory cytokine IL-10 and/or TGF-β. Furthermore, it is noticeable that Tregs likely contribute to allergic disorders such as dermatitis and airway inflammation, and play a crucial role in the treatment of allergy through their actions on suppression of effector T cells and inhibition of activation of mast cells and basophils. Modulation of functions of Tregs may provide a novel strategy to prevent and treat allergic diseases. PMID:24886492

  5. 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.

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

    Science.gov (United States)

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

    2015-02-01

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

  7. Role of Exosome Shuttle RNA in Cell-to-Cell Communication.

    Science.gov (United States)

    Zhang, Wei; Peng, Peng; Shen, Keng

    2016-08-01

    There are several ways that transpire in cell-to-cell communication,with or without cell contact. Exosomes play an important role in cell-to-cell communication,which do not need cell contact,as that can result in a relatively long-distance influence. Exosome contains RNA components including mRNA and micro-RNA,which are protected by exosomes rigid membranes. This allows those components be passed long distance through the circulatory system. The mRNA components are far different from their donor cells,and the micro-RNA components may reflect the cell they originated. In this article we review the role of exosomes in cell-to-cell communication,with particular focus on their potentials in both diagnostic and therapeutic applications. PMID:27594165

  8. Comparative expression profiling reveals a role of the root apoplast in local phosphate response

    OpenAIRE

    Hoehenwarter, Wolfgang; Mönchgesang, Susann; Neumann, Steffen; Majovsky, Petra; Abel, Steffen; Jens MÜLLER

    2016-01-01

    Background Plant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacti...

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

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Xuewei Wang

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

  11. Regulatory T Cells and Their Role in Animal Disease.

    Science.gov (United States)

    Veiga-Parga, T

    2016-07-01

    In humans and mouse models, Foxp3(+) regulatory T cells are known to control all aspects of immune responses. However, only limited information exists on these cells' role in diseases of other animals. In this review, we cover the most important features and different types of regulatory T cells, which include those that are thymus-derived and peripherally induced, the mechanisms by which they control immune responses by targeting effector T cells and antigen-presenting cells, and most important, their role in animal health and diseases including cancer, infections, and other conditions such as hypersensitivities and autoimmunity. Although the literature regarding regulatory T cells in domestic animal species is still limited, multiple articles have recently emerged and are discussed. Moreover, we also discuss the evidence suggesting that regulatory T cells might limit the magnitude of effector responses, which can have either a positive or negative result, depending on the context of animal and human disease. In addition, the issue of plasticity is discussed because plasticity in regulatory T cells can result in the loss of their protective function in some microenvironments during disease. Lastly, the manipulation of regulatory T cells is discussed in assessing the possibility of their use as a treatment in the future. PMID:26945003

  12. Prodigiosin inhibits motility and activates bacterial cell death revealing molecular biomarkers of programmed cell death.

    Science.gov (United States)

    Darshan, N; Manonmani, H K

    2016-12-01

    The antimicrobial activity of prodigiosin from Serratia nematodiphila darsh1, a bacterial pigment was tested against few food borne bacterial pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The mode of action of prodigiosin was studied. Prodigiosin induced bactericidal activity indicating a stereotypical set of biochemical and morphological feature of Programmed cell death (PCD). PCD involves DNA fragmentation, generation of ROS, and expression of a protein with caspase-like substrate specificity in bacterial cells. Prodigiosin was observed to be internalized into bacterial cells and was localized predominantly in the membrane and the nuclear fraction, thus, facilitating intracellular trafficking and then binding of prodigiosin to the bacterial DNA. Corresponding to an increasing concentration of prodigiosin, the level of certain proteases were observed to increase in bacteria studied, thus initiating the onset of PCD. Prodigiosin at a sub-inhibitory concentration inhibits motility of pathogens. Our observations indicated that prodigiosin could be a promising antibacterial agent and could be used in the prevention of bacterial infections. PMID:27460563

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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

  15. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. 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.

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

    Science.gov (United States)

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

    2016-09-01

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

  20. 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.

  1. Role of pancreatic stellate cells in chemoresistance in pancreatic cancer

    OpenAIRE

    McCarroll, Joshua A.; Naim, Stephanie; Sharbeen, George; Russia, Nelson; Lee, Julia; Kavallaris, Maria; Goldstein, David; Phillips, Phoebe A.

    2014-01-01

    Pancreatic cancer is highly chemoresistant. A major contributing factor is the characteristic extensive stromal or fibrotic reaction, which comprises up to 90% of the tumor volume. Over the last decade there has been intensive research into the role of the pro-fibrogenic pancreatic stellate cells (PSCs) and their interaction with pancreatic cancer cells. As a result of the significant alterations in the tumor microenvironment following activation of PSCs, tumor progression, and chemoresistanc...

  2. 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

  3. 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...

  4. The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation.

    Science.gov (United States)

    Rodor, Julie; Pan, Qun; Blencowe, Benjamin J; Eyras, Eduardo; Cáceres, Javier F

    2016-09-01

    Acinus (apoptotic chromatin condensation inducer in the nucleus) is an RNA-binding protein (RBP) originally identified for its role in apoptosis. It was later found to be an auxiliary component of the exon junction complex (EJC), which is deposited at exon junctions as a consequence of pre-mRNA splicing. To uncover the cellular functions of Acinus and investigate its role in splicing, we mapped its endogenous RNA targets using the cross-linking immunoprecipitation protocol (iCLIP). We observed that Acinus binds to pre-mRNAs, associating specifically to a subset of suboptimal introns, but also to spliced mRNAs. We also confirmed the presence of Acinus as a peripheral factor of the EJC. RNA-seq was used to investigate changes in gene expression and alternative splicing following siRNA-mediated depletion of Acinus in HeLa cells. This analysis revealed that Acinus is preferentially required for the inclusion of specific alternative cassette exons and also controls the faithful splicing of a subset of introns. Moreover, a large number of splicing changes can be related to Acinus binding, suggesting a direct role of Acinus in exon and intron definition. In particular, Acinus regulates the splicing of DFFA/ICAD transcript, a major regulator of DNA fragmentation. Globally, the genome-wide identification of RNA targets of Acinus revealed its role in splicing regulation as well as its involvement in other cellular pathways, including cell cycle progression. Altogether, this study uncovers new cellular functions of an RBP transiently associated with the EJC. PMID:27365209

  5. 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.

  6. 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.

  7. 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. PMID:26780491

  8. 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....

  9. The military and domestic disaster response lead role revealed through the eye of Hurricane Katrina?

    OpenAIRE

    Walker, Juliana M.

    2006-01-01

    The traditional role of the active-duty military force at home is one of support to a civilian Lead Federal Agency (LFA) that primarily falls under the Department of Homeland Security (DHS). During emergencies, military domestic assistance is historically provided when local, state, and federal resources have been overwhelmed. During and in the aftermath of Hurricane Katrina, however, the slow and perceived inept response to the massive disaster prompted a national debate on the appropriate...

  10. Quadruple zebrafish mutant reveals different roles of Mesp genes in somite segmentation between mouse and zebrafish.

    Science.gov (United States)

    Yabe, Taijiro; Hoshijima, Kazuyuki; Yamamoto, Takashi; Takada, Shinji

    2016-08-01

    The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists, especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays crucial roles in this conversion in the mouse. However, it remains unclear whether Mesp genes play similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all four zebrafish Mesp genes by using TALEN-mediated genome editing. Contrary to the situation in the mouse Mesp2 mutant, in the zebrafish Mesp quadruple mutant embryos the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. However, each somite was caudalized in a similar manner to the mouse Mesp2 mutant, and the superficial horizontal myoseptum and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the link between the molecular clock and somite morphogenesis. PMID:27385009

  11. Experimental taphonomy of Artemia reveals the role of endogenous microbes in mediating decay and fossilization.

    Science.gov (United States)

    Butler, Aodhán D; Cunningham, John A; Budd, Graham E; Donoghue, Philip C J

    2015-06-01

    Exceptionally preserved fossils provide major insights into the evolutionary history of life. Microbial activity is thought to play a pivotal role in both the decay of organisms and the preservation of soft tissue in the fossil record, though this has been the subject of very little experimental investigation. To remedy this, we undertook an experimental study of the decay of the brine shrimp Artemia, examining the roles of autolysis, microbial activity, oxygen diffusion and reducing conditions. Our findings indicate that endogenous gut bacteria are the main factor controlling decay. Following gut wall rupture, but prior to cuticle failure, gut-derived microbes spread into the body cavity, consuming tissues and forming biofilms capable of mediating authigenic mineralization, that pseudomorph tissues and structures such as limbs and the haemocoel. These observations explain patterns observed in exceptionally preserved fossil arthropods. For example, guts are preserved relatively frequently, while preservation of other internal anatomy is rare. They also suggest that gut-derived microbes play a key role in the preservation of internal anatomy and that differential preservation between exceptional deposits might be because of factors that control autolysis and microbial activity. The findings also suggest that the evolution of a through gut and its bacterial microflora increased the potential for exceptional fossil preservation in bilaterians, providing one explanation for the extreme rarity of internal preservation in those animals that lack a through gut. PMID:25972468

  12. Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

    Science.gov (United States)

    Tebaldi, Giulia; Jacca, Sarah; Montanini, Barbara; Capra, Emanuele; Rosamilia, Alfonso; Sala, Arianna; Stella, Alessandra; Castiglioni, Bianca; Ottonello, Simone; Donofrio, Gaetano

    2016-07-01

    Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation. PMID:27281703

  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. 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.

  15. 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

  16. 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

  17. Role of Mitochondrial Translocation of Telomerase in Hepatocellular Carcinoma Cells with Multidrug Resistance

    Directory of Open Access Journals (Sweden)

    Xianlong Ling, Lei Wen, Yuan Zhou

    2012-01-01

    Full Text Available Multidrug resistance (MDR is a major obstacle of cancer chemotherapy. This study aimed to investigate the role of mitochondrial translocation of telomerase (hTERT in MDR of human hepatocellular carcinoma (HCC cells. In this study, three HCC cell lines (SK-Hep1/CDDP1 cells, SK-Hep1/CDDP2 cells and SK-Hep1/CDDP3 cells with differential resistance index (RI to cisplatin (CDDP were induced by pulse treatment of SK-Hep1 (human hepatocellular cell line with CDDP in vitro. The RI of SK-Hep1/CDDP1 cells, SK-Hep1/CDDP2 cells and SK-Hep1/CDDP3 cells was 5.14, 8.66, and 14.25, respectively, and all the cell lines showed cross-resistance to Doxorubicin (DOX and 5-Fuorouracil (5-FU. The apoptosis rates in drug-resistant cells were significantly reduced. Cell cycle analysis revealed the ratio of drug-resistant cells in G2/M and S phases increased, while that in G1 phase decreased. Immunofluorescence staining and Western blot assay demonstrated, with the gradual elevation in RI, increasing hTERT translocated from the nuclei to the mitochondria, while real-time PCR indicated the shortening of telomere length in drug-resistant cells under the chemotherapeutic stress and the reduction of damaged mtDNA with the increase in RI. Furthermore, JC-1 staining also indicated the reduction of mitochondrial membrane potential in drug-resistant cells. The mitochondrial translocation of hTERT increases in multidrug-resistant cells and exerts protective effect on mitochondrial function. Drug-resistant tumor cells escape from apoptosis through hTERT-mediated mitochondrial protection. Mitochondrial translocation of hTERT may serve as an underlying mechanism of MDR.

  18. Role of mitochondrial translocation of telomerase in hepatocellular carcinoma cells with multidrug resistance.

    Science.gov (United States)

    Ling, Xianlong; Wen, Lei; Zhou, Yuan

    2012-01-01

    Multidrug resistance (MDR) is a major obstacle of cancer chemotherapy. This study aimed to investigate the role of mitochondrial translocation of telomerase (hTERT) in MDR of human hepatocellular carcinoma (HCC) cells. In this study, three HCC cell lines (SK-Hep1/CDDP1 cells, SK-Hep1/CDDP2 cells and SK-Hep1/CDDP3 cells) with differential resistance index (RI) to cisplatin (CDDP) were induced by pulse treatment of SK-Hep1 (human hepatocellular cell line) with CDDP in vitro. The RI of SK-Hep1/CDDP1 cells, SK-Hep1/CDDP2 cells and SK-Hep1/CDDP3 cells was 5.14, 8.66, and 14.25, respectively, and all the cell lines showed cross-resistance to Doxorubicin (DOX) and 5-Fuorouracil (5-FU). The apoptosis rates in drug-resistant cells were significantly reduced. Cell cycle analysis revealed the ratio of drug-resistant cells in G2/M and S phases increased, while that in G1 phase decreased. Immunofluorescence staining and Western blot assay demonstrated, with the gradual elevation in RI, increasing hTERT translocated from the nuclei to the mitochondria, while real-time PCR indicated the shortening of telomere length in drug-resistant cells under the chemotherapeutic stress and the reduction of damaged mtDNA with the increase in RI. Furthermore, JC-1 staining also indicated the reduction of mitochondrial membrane potential in drug-resistant cells. The mitochondrial translocation of hTERT increases in multidrug-resistant cells and exerts protective effect on mitochondrial function. Drug-resistant tumor cells escape from apoptosis through hTERT-mediated mitochondrial protection. Mitochondrial translocation of hTERT may serve as an underlying mechanism of MDR. PMID:22991493

  19. 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.

  20. 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.

  1. Role of Ikaros in T-cell acute lymphoblastic leukemia

    OpenAIRE

    Kastner, Philippe; Chan, Susan

    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 l...

  2. Role of plasmacytoid dendritic cell subsets in allergic asthma

    OpenAIRE

    Maazi, Hadi; Lam, Jonathan; Lombardi, Vincent; Akbari, Omid

    2013-01-01

    Plasmacytoid dendritic cells (pDCs) are major type-I interferon producing cells that play important roles in antiviral immunity and tolerance induction. These cells share a common DC progenitor with conventional DCs and Fms-like tyrosine kinase-3 ligand is essential for their development. Several subsets of pDCs have been identified to date including CCR9+, CD9+ and CD2+ pDCs. Recently, three subsets of pDCs were described namely, CD8α−β−, CD8α+β− and CD8α+β+ subsets. Interestingly, CD8α+β− a...

  3. 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

  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. The role of physical forces on cytotoxic T cell-target cell conjugate stability.

    Science.gov (United States)

    Hubbard, B B; Glacken, M W; Rodgers, J R; Rich, R R

    1990-06-01

    Theoretical considerations suggest that external forces play a significant role in cell-cell conjugate formation and may lead to the misinterpretation of adhesion data. To test this, the stability of conjugates formed between CTL and fibroblast target cells (TC) was examined in the controlled shear environment of a parallel plate flow chamber. Murine fibroblast targets expressing class I maternally transmitted Ag Mtaa or Mtab were grown on a glass slide that formed one wall of the flow chamber and were used in conjunction with anti-Mtaa and anti-Mtab specific mouse CTL clones to establish a panel of Ag-reciprocal targets and lymphocytes. Although cytolysis assays indicated that lymphocytes recognized and destroyed appropriate but not inappropriate targets, the stability of some CTL/TC conjugates was Ag independent. In all cases, the conjugate stability was shear dependent over a 100-fold range (0.04 to 4.0 dynes/cm2). For some clones, the ratio of the stabilities of Ag-specific CTL/TC conjugates to nonspecific conjugates was significantly enhanced with increasing shear. This implies that the role of Ag specificity in CTL/TC adhesion may be misinterpreted if the shear environment of CTL/TC conjugates is unknown or uncontrolled. Kinetic analysis revealed that conjugate stability was dependent on the exposure time to external forces and that there existed two populations of conjugates; weak associations that disengaged within the first 30 s of flow, and strong associations that remained attached even after a 5-min exposure to a steady shear stress. The stability of Ag-specific CTL/TC conjugates at 0.04 dynes/cm2 was enhanced by 50% as the temperature was increased from 25 to 37 degrees C, whereas the stability of nonspecific CTL/TC associations was not affected. This result indicates that significant Ag-specific strengthening may occur at physiologic temperatures. This work suggests the importance of attention to role of fluid mechanical shear stress in standard

  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. The Role of Plant Hormones in Nematode Feeding Cell Formation

    NARCIS (Netherlands)

    Goverse, A.; Bird, D.

    2011-01-01

    In this Chapter, we discuss recent advances in the role of plant hormones in the molecular mechanisms underlying feeding cell formation both by cyst (CN) and root-knot nematodes (RKN). Phytohormones are small signalling molecules that regulate plant growth and development, including the formation of

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

    Science.gov (United States)

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

    2016-01-01

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

  9. 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.

  10. Population transcriptomics reveals a potentially positive role of expression diversity in adaptation

    Institute of Scientific and Technical Information of China (English)

    Qin Xu; Fei Shang; Lifang Kang; Wenli Chen; Juan Yan; Jianqiang Li; Tao Sang; Shilai Xing; Caiyun Zhu; Wei Liu; Yangyang Fan; Qian Wang; Zhihong Song; Wenhui Yang; Fan Luo

    2015-01-01

    While it is widely accepted that genetic diversity determines the potential of adaptation, the role that gene expression variation plays in adaptation remains poorly known. Here we show that gene expression diversity could have played a positive role in the adaptation of Miscanthus lutarioriparius. RNA‐seq was conducted for 80 individuals of the species, with half planted in the energy crop domestication site and the other half planted in the control site near native habitats. A leaf reference transcriptome consisting of 18,503 high‐quality tran-scripts was obtained using a pipeline developed for de novo assembling with population RNA‐seq data. The population structure and genetic diversity of M. lutarioriparius were estimated based on 30,609 genic single nucleotide polymor-phisms. Population expression (Ep) and expression diversity (Ed) were defined to measure the average level and the magnitude of variation of a gene expression in the population, respectively. It was found that expression diversity increased while genetic diversity decreased after the species was transplanted from the native habitats to the harsh domestication site, especial y for genes involved in abiotic stress resistance, histone methylation, and biomass synthesis under water limitation. The increased expression diversity could have enriched phenotypic variation directly subject to selections in the new environment.

  11. 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.

  12. 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

  13. 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.

  14. 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

    2007-10-01

    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. PMID:17913879

  15. 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

  16. 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

  17. The putative role of mast cells in lung transplantation.

    Science.gov (United States)

    Jungraithmayr, W

    2015-03-01

    Mast cells (MCs) were primarily recognized as effector cells of allergy. These cells are acting predominantly at the interface between the host and the external environment, such as skin, gastrointestinal and the respiratory tract. Only recently, MCs have gained increased recognition as cells of functional plasticity with immune-regulatory properties that influence both the innate and the adaptive immune response in inflammatory disorders, cancer and transplantation. Through the secretion of both proinflammatory and antiinflammatory mediators, MCs can either ameliorate or deteriorate the course and outcome in lung transplantation. Recent research from other models recognized the immune-protective activity of MCs including its role as an important source of IL-10 and TGF-β for the modulation of alloreactive T cell responses or assistance in Treg activity. This paper summarizes the current understanding of MCs in lung transplantation and discusses MC-mediated immune-mechanisms by which the outcome of the engrafted organ is modulated. PMID:25693471

  18. 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.

  19. 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.

  20. [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. PMID:8786892

  1. [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.

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

    OpenAIRE

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

    2007-01-01

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

  3. 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.

  4. 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

  5. Role of calcium in differentiation of murine erythroleukemia cells

    Institute of Scientific and Technical Information of China (English)

    ZHUDAN; NONGGAOHE; 等

    1993-01-01

    Calcium plays a crucial role in the normal and abnomal cell metabolism.The role of calcium in the differentiation process of murine erythroleukemia cells(MELC)remains controversial.Here,based upon quantitative measurement of fluorescence in single cells,a method was developed to investigate the intracellular free calcium[Ca2+]i concentration and DNA contents simultaneously,by employing the fluorescent probe,fluo-3 acetoxymethyl ester and DNA dye Hoechst 33342.During MELC differentiation.[Ca2+]i concentration incresed.We also demonstrated that calcium ionophore,A23187,enhanced the HMB-induced MELC differentiation,while verapamil,an inhibitor of calcuim uptake,slightly reduced differentiation.These results suggested that an increase in the [Ca2+]i level was an essential step in HMBA-induced MELC differentiation.

  6. 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.

  7. 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.

  8. 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

  9. Real-Time Imaging of Resident T Cells in Human Lung and Ovarian Carcinomas Reveals How Different Tumor Microenvironments Control T Lymphocyte Migration.

    Science.gov (United States)

    Bougherara, Houcine; Mansuet-Lupo, Audrey; Alifano, Marco; Ngô, Charlotte; Damotte, Diane; Le Frère-Belda, Marie-Aude; Donnadieu, Emmanuel; Peranzoni, Elisa

    2015-01-01

    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 contacts 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 the control of tumor growth. This approach will aid in the development and testing of novel immunotherapy strategies to promote T cell migration in

  10. 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.

  11. Continental cichlid radiations: functional diversity reveals the role of changing ecological opportunity in the Neotropics.

    Science.gov (United States)

    Arbour, Jessica Hilary; López-Fernández, Hernán

    2016-08-17

    Adaptive radiations have been hypothesized to contribute broadly to the diversity of organisms. Models of adaptive radiation predict that ecological opportunity and ecological release, the availability of empty ecological niches and the response by adapting lineages to occupy them, respectively, drive patterns of phenotypic and lineage diversification. Adaptive radiations driven by 'ecological opportunity' are well established in island systems; it is less clear if ecological opportunity influences continent-wide diversification. We use Neotropical cichlid fishes to test if variation in rates of functional evolution is consistent with changing ecological opportunity. Across a functional morphological axis associated with ram-suction feeding traits, evolutionary rates declined through time as lineages diversified in South America. Evolutionary rates of ram-suction functional morphology also appear to have accelerated as cichlids colonized Central America and encountered renewed opportunity. Our results suggest that ecological opportunity may play an important role in shaping patterns of morphological diversity of even broadly distributed lineages like Neotropical cichlids. PMID:27512144

  12. 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.

  13. Pair dispersion in a chaotic flow reveals the role of the memory of initial velocity

    CERN Document Server

    Afik, Eldad

    2015-01-01

    The leading paradigm for chaotic flows dominated by dissipation predicts an exponential growth of the mean distance between pairs of fluid elements, in the long run. This is reflected in the analysis of experimental results on tracer particles and the discussions which follow, as reported in recent experimental and numerical publications. To quantitatively validate this prediction, we have conducted a microfluidic experiment generating elastic turbulence, a flow characterised in the literature as smooth in space and random in time. To our great surprise, we discovered that the pair separation follows a much slower power-law --- also known as ballistic --- a notion overlooked so far for flows of this type. We provide conclusive experimental evidence that this scaling is well-desribed by the same coefficients derived from the short-time dynamics. Our finding reinforces the role of the ballistic regime over a significant range in time and space, providing a quantitative estimation for the spreading of particles ...

  14. 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.

  15. 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.

  16. 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.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2011-11-01

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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. KCNQ Modulators Reveal a Key Role for KCNQ Potassium Channels in Regulating the Tone of Rat Pulmonary Artery Smooth Muscle

    OpenAIRE

    Joshi, Shreena; Sedivy, Vojtech; Hodyc, Daniel; Herget, Jan; Gurney, Alison M

    2009-01-01

    Potassium channels are central to the regulation of pulmonary vascular tone. The smooth muscle cells of pulmonary artery display a background K+ conductance with biophysical properties resembling those of KCNQ (KV7) potassium channels. Therefore, we investigated the expression and functional role of KCNQ channels in pulmonary artery. The effects of selective KCNQ channel modulators were investigated on K+ current and membrane potential in isolated pulmonary artery smoo...

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

    Science.gov (United States)

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

    2016-01-01

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

  5. 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.

  6. 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.

  7. 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...

  8. 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.

  9. 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.

  10. Community Lenses Revealing the Role of Sociocultural Environment on Physical Activity

    Science.gov (United States)

    Belon, Ana Paula; Nieuwendyk, Laura M.; Vallianatos, Helen; Nykiforuk, Candace I. J.

    2016-01-01

    Purpose To identify perceptions of how sociocultural environment enabled and hindered physical activity (PA) participation. Design Community-based participatory research. Setting Two semirural and two urban communities located in Alberta, Canada. Participants Thirty-five people (74.3% females, 71.4% aged 25–64 years) across the four communities. Method PhotoVoice activities occurred over 3 months during the spring of 2009. Participants were asked to document perceived environmental attributes that might foster or inhibit PA in their community. Photographs and narratives were shared in one-on-one interviews. Line-by-line coding of the transcripts was independently conducted by two researchers using an inductive approach. Codes were arranged into themes and subthemes, which were then organized into the Analysis Grid for Environments Linked to Obesity (ANGELO) framework. Results Six main themes (accompanied by subthemes) emerged: sociocultural aesthetics, safety, social involvement, PA motivation, cultural ideas of recreation, and car culture. Representative quotes and photographs illustrate enablers and obstacles identified by participants. Conclusion This PhotoVoice study revealed how aspects of participants’ sociocultural environments shaped their decisions to be physically active. Providing more PA resources is only one step in the promotion of supportive environments. Strategies should also account for the beautification and maintenance of communities, increasing feelings of safety, enhancement of social support among community members, popularization of PA, and mitigating car culture, among others. PMID:25973966

  11. 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. PMID:26646505

  12. The role of interfaces in organic spin valves revealed through spectroscopic and transport measurements

    Energy Technology Data Exchange (ETDEWEB)

    Drew, Alan J.; Nuccio, Laura; Gillin, William P. [School of Physics, Queen Mary University of London, Mile End Road, E1 4NS London (United Kingdom); Szulczewski, Gregory [Department of Chemistry, University of Alabama, Box 870336, Tuscaloosa, AL 35487-0336 (United States)

    2012-01-15

    Interfaces between dissimilar materials are critical to the performance of many electronic devices. In electrical devices where the active transport is through an organic semiconductor (OSC) layer there are metallic electrodes to supply and remove the charge carriers. At metal/molecule and molecule/metal interfaces the electrical contact is usually not Ohmic. As a result, in most metal-organic semiconductors-metal devices charge injection is mainly governed by tunneling across the barriers. When the metal electrodes are ferromagnetic the density of occupied and unoccupied electronic states is spin-dependent, which presents further subtleties to the electron transfer. In this article we summarize some of the important experimental results that have advanced our understanding of spin-polarized electron transfer across ferromagnetic metal (FM)/OSC interfaces. In particular, we highlight key spectroscopic studies that have revealed insights into the nature of the electronic structure between OSCs and FMs, in particular between Alq{sub 3} and Co and Fe surfaces. We discuss the relationship between energy level diagrams for these systems and transport measurements made on spin-valve devices. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Liang-Hui Chu

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

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

    Science.gov (United States)

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

    2012-07-01

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

  16. Potential primary roles of glial cells in the mechanisms of psychiatric disorders

    Directory of Open Access Journals (Sweden)

    Kazuhiko eYamamuro

    2015-05-01

    Full Text Available 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.

  17. 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. PMID:26029044

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    Directory of Open Access Journals (Sweden)

    Robert Kalescky

    2016-04-01

    Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  6. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    Science.gov (United States)

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

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

    Science.gov (United States)

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

    2014-10-01

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

  8. Prenatal tolerance--a role for regulatory T cells?

    Science.gov (United States)

    Izcue, Ana; Powrie, Fiona

    2005-02-01

    Regulatory T cells (TR cells) play a major role in controlling immune self reactivity. However, little is known about their occurrence and functions in early developmental stages. In this issue of the European Journal of Immunology, Cupedo et al. report the presence of functional CD4+CD25+ TR cells in the human fetus. In contrast to previous studies, the analysis is performed on fetal thymus, spleen and lymph node samples in addition to cord blood cells. Interestingly, TR cells are present in all these organs from 14 weeks of gestation, along with FoxP3 (forkhead box protein 3) RNA, a marker for naturally arising TR cells. The fetal TR cells show, however, phenotypic differences depending on their location, possibly because of variations in their activation state. The emergence of TR cells so early in fetal development raises a number of questions about the mechanisms of self reactivity and tolerance in the prenatal stages, which may have important implications for our understanding of childhood pathologies. PMID:15682452

  9. 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...

  10. 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.

  11. 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.

  12. 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.

  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. Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

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    Roberto Pérez-Torrado

    Full Text Available In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

  15. 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.

  16. 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.

  17. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

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    Holger A. Russ

    2016-01-01

    Full Text Available Current approaches in human embryonic stem cell (hESC to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1, Neuroplastin (NPTN, and the Laminin α-2 subunit (LAMA2. Two of the three factors (LGALS1 and LAMA2 increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

  18. 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.

  19. 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.

  20. 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.

  1. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

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    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

  2. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  3. A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

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    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.

  4. 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.

  5. 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 cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology. CONCLUSIONS: These data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.

  6. 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...

  7. Role of Plasmodium falciparum thrombospondin-related anonymous protein in host-cell interactions

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    Malhotra Pawan

    2008-04-01

    Full Text Available Abstract Background Thrombospondin-related anonymous protein (TRAP is essential for sporozoite motility and for liver cell invasion. TRAP is a type 1 membrane protein that possesses multiple adhesive domains in its extracellular region. Methods Plasmodium falciparum TRAP (PfTRAP and its subdomains were expressed in a mammalian expression system, and eleven different mutants generated to study interaction of PfTRAP with liver cells. Binding studies between HepG2 cell extracts and PfTRAP were performed using co-immunoprecipitation protocols. Results Five different amino acid residues of PfTRAP that are involved in liver cell binding have been identified. These PfTRAP mutants bound to heparin like the wild type PfTRAP thereby suggesting a non-heparin mediated binding of PfTRAP to liver cells. Three Src family proteins -Lyn, Lck and CrkL which interact with PfTRAP are also identified. Liver cell extracts and immunoprecipitated Src family kinases phosphorylated PfTRAP at multiple sites. An analysis of multiple TRAP sequences revealed Src homology 3 domain (SH3 binding motifs. Conclusion Binding of PfTRAP to SH3-domain containing proteins like Src-family kinases and their ability to phosphorylate PfTRAP suggests a novel role for PfTRAP in cell signaling during sporozoite invasion and homing inside the liver cells. These data shed new light on TRAP-liver cell interactions.

  8. Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells.

    Science.gov (United States)

    Castoria, G; Giovannelli, P; Di Donato, M; Ciociola, A; Hayashi, R; Bernal, F; Appella, E; Auricchio, F; Migliaccio, A

    2014-12-04

    The functions of androgen receptor (AR) in stromal cells are still debated in spite of the demonstrated importance of these cells in organ development and diseases. Here, we show that physiological androgen concentration (10 nM R1881 or DHT) fails to induce DNA synthesis, while it consistently stimulates cell migration in mesenchymal and transformed mesenchymal cells. Ten nanomolar R1881 triggers p27 Ser10 phosphorylation and its stabilization in NIH3T3 fibroblasts. Activation of Rac and its downstream effector DYRK 1B is responsible for p27 Ser10 phosphorylation and cell quiescence. Ten nanomolar androgen also inhibits transformation induced by oncogenic Ras in NIH3T3 fibroblasts. Overexpression of an AR mutant unable to interact with filamin A, use of a small peptide displacing AR/filamin A interaction, and filamin A knockdown indicate that the androgen-triggered AR/filamin A complex regulates the pathway leading to p27 Ser10 phosphorylation and cell cycle arrest. As the AR/filamin A complex is also responsible for migration stimulated by 10 nM androgen, our report shows that the androgen-triggered AR/filamin A complex controls, through Rac 1, the decision of cells to halt cell cycle and migration. This study reveals a new and unexpected role of androgen/AR signalling in coordinating stromal cell functions.

  9. 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.

  10. 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.

  11. Cardiac Metastases of Renal Cell Carcinoma Revealed by Syncope: Diagnosis and Treatment

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    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.

  12. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

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    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Role of gamma-delta T cells in liver inflammation and fibrosis

    Institute of Scientific and Technical Information of China (English)

    Linda; Hammerich; Frank; Tacke

    2014-01-01

    Conventional adaptive T cell responses contribute to liver inflammation and fibrogenesis,especially in chronic viral infections and autoimmune hepatitis.However,the role of unconventional gamma-delta(γδ)T cells in liver diseases is less clear.In the past two decades,accumulating evidence revealed thatγδT cell numbers remarkably increase in the liver upon various inflammatory conditions in mice and humans.More recent studies demonstrated that the functional effect ofγδT cells on liver disease progression depends on the subsets involved,which can be identified by the expression of distinct T cell receptor chains and of specific cytokines.Fascinatingly,γδT cells may have protective as well as pathogenic functions in liver diseases.Interferonγ-producingγδT cells,for example,induce apoptosis in hepatocytes but also in hepatic tumor cells;while interleukin-17-expressingγδT cells can downregulate pathogenic effector functions of other immune cells and can promote apoptosis of fibrogenic stellate cells.However,the results obtained in human liver disease as well as murine models are not fully conclusive at present,and the effects ofγδT cells on the outcome of liver disease might vary dependent on etiology and stage of disease.Further definitions of theγδT cell subsets in-volved in acute and chronic liver inflammation,as well as their effector cytokines might uncover whether interference withγδT cells could be a useful target for the treatment of liver disease.

  15. Inhibitory Role of Pentraxin-3 in Esophageal Squamous Cell Carcinoma

    Science.gov (United States)

    Ma, Dan; Zong, Ye; Zhu, Sheng-Tao; Wang, Yong-Jun; Li, Peng; Zhang, Shu-Tian

    2016-01-01

    Background: Esophageal cancer is the sixth leading cause of cancer-related death worldwide. Pentraxin-3 (PTX3) is a member of the PTX superfamily. Here, we investigated the role of PTX3 in esophageal squamous cell carcinoma (ESCC). Methods: The effect of PTX3 on ESCC cell proliferation, colony formation, apoptosis, migration, and invasion was investigated using cell viability assays, colony formation assays, flow cytometry, and migration and invasion assays. The effect of PTX3 on the tumorigenicity of ESCC in vivo was investigated with xenograft studies in nude mice. Results: PTX3 overexpression in ESCC cells reduced cellular proliferation and colony formation (P apoptosis (P < 0.05). PTX3 expression had no significant effect on the migratory or invasive potential of ESCC cells. In our mouse model of human ESCC, we achieved 100% successful tumor establishment. Compared with the control and empty vector-expressing groups, the PTX3-expressing group formed significantly smaller tumors (P < 0.05). Conclusions: This study indicates that PTX3 might play an inhibitory role in ESCC. PMID:27625097

  16. Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jian-Min Su; Li-Ying Wang; Yu-Long Liang; Xi-Liang Zha

    2005-01-01

    AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase)ILK, and β-catenin in hepatocellular carcinoma cell apoptosis.METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and 1H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 μmol/L N,N-diphenyl-p-phenylenediamine,which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, β-catenin, and PKB in this apoptotic model by Western blot.RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and β-catenin or the damage of cell-matrix and cell-cell adhesion.CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and β-catenin,could induce hepatocellular carcinoma cell apoptosis.

  17. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Directory of Open Access Journals (Sweden)

    Shufeng Liu

    Full Text Available Hepatitis C virus (HCV poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP, UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1 and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.

  18. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Science.gov (United States)

    Liu, Shufeng; Zhao, Ting; Song, BenBen; Zhou, Jianhua; Wang, Tony T

    2016-01-01

    Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. PMID:26808496

  19. Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing

    Directory of Open Access Journals (Sweden)

    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.

  20. The Role of Myeloma Cells to Osteoclast Activation

    Directory of Open Access Journals (Sweden)

    Bahare Sadeghi

    2010-01-01

    Full Text Available Objective: Multiple myeloma (MM is a hematological malignancy characterized by osteolyticbone disease which is associated with severe bone pain and pathological bonefractures. The receptor activator of nuclear factor κB (RANK and receptor activator ofnuclear factor κB ligand (RANKL system has an important role in regulation of boneremodeling process. The aim of this study was to evaluate the expression of the RANK/RANKL molecules by the myeloma cells derived from patients and myeloma cell lineU-266.Materials and Methods: Myeloma cells derived from 7 myeloma patients and plasma cellleukemia were included into this study to evaluate the expression of the RANK/RANKLmolecules by the reverse transcriptions-polymerase chain reaction (RT-PCR method atthe mRNA level. As well as human myeloma cell line U266, U937, RPMI-8866 and Helawere used as control groups.Results: In this study we show the expression of RANK and its ligand at the mRNA levelin U-266 (myeloma cell line and plasma cells derived from patients by the RT-PCR technique.Conclusion: Our results demonstrate that expression of RANK and RANKL by plasmacells can contribute to induction of osteoclasts and plasma cell activation which elevatesbone resorption in myeloma patients.

  1. Role of Inflammation and Substrate Stiffness in Cancer Cell Transmigration

    Science.gov (United States)

    Hamilla, Susan; Stroka, Kimberly; Aranda-Espinoza, Helim

    2013-03-01

    Cancer metastasis, the ability for cancer cells to break away from the primary tumor site and spread to other organs of the body, is one of the main contributing factors to the deadliness of the disease. One of the rate-limiting steps in cancer metastasis that is not well understood is the adhesion of tumor cells to the endothelium followed by transmigration. Other factors include substrate stiffness and inflammation. To test these parameters, we designed an in vitro model of transendothelial migration. Our results suggest that cancer cell transmigration is a two-step process in which they first incorporate into the endothelium before migrating through. It was observed that the cumulative fraction of cancer cells that incorporate into the endothelium increases over time. Unlike leukocytes, which can directly transmigrate through the endothelium, cancer cells appear to have a two-step process of transmigration. Our results indicate that inflammation does not act as a signal for cancer cells to localize at specific sites and transmigrate similarly to leukocytes. Cancer cell transmigration also does not vary with substrate stiffness indicating that tissue stiffness may not play a role in cancer's propensity to metastasize to certain tissues.

  2. Role of mast cells in gastrointestinal mucosal defense.

    Science.gov (United States)

    Penissi, Alicia B; Rudolph, María I; Piezzi, Ramón S

    2003-08-01

    The purpose of this review, based on studies from our laboratory as well as from others, is to summarize salient features of mast cell immunobiology and to describe their associations with gastrointestinal mucosal defense. Gastrointestinal mast cells are involved in many pathologic effects, such as food hypersensitivity. On the other hand, they also play a protective role in defense against parasitic and microbial infections. Thus, they have both positive and negative effects, but presently the mechanisms that control the balance of these various effects are poorly known. It has been suggested that stabilization of mast cells may be a key mechanism to protect the gastrointestinal tract from injury. Few molecules are known to possess both mast cell stabilizing and gastrointestinal cytoprotective activity. These include zinc compounds, sodium cromoglycate, FPL 52694, ketotifen, aloe vera, certain flavonoids such as quercetin, some sulfated proteoglycans such as chondroitin sulfate and dehydroleucodine. Dehydroleucodine, a sesquiterpene lactone isolated from Artemisia douglasiana Besser, exhibits anti-inflammatory and gastrointestinal cytoprotective action. The lactone stimulates mucus production, and inhibits histamine and serotonin release from intestinal mast cells. The lactone could act as a selective mast cell stabilizer by releasing cytoprotective factors and inhibiting pro-inflammatory mast cell mediators. PMID:14510234

  3. Role of Natural Radiosensitizers and Cancer Cell Radioresistance: An Update

    Directory of Open Access Journals (Sweden)

    Arif Malik

    2016-01-01

    Full Text Available Cancer originates from genetic mutations accumulation. Cancer stem cells have been depicted as tumorigenic cells that can differentiate and self-renew. Cancer stem cells are thought to be resistant to conventional therapy like chemotherapy and radiation therapy. Radiation therapy and chemotherapy damage carcinomic DNA cells. Because of the ability of cancer stem cells to self-renew and reproduce malignant tumors, they are the subject of intensive research. In this review, CSCs radioresistant mechanisms which include DNA damage response and natural radiosensitizers have been summed up. Reactive oxygen species play an important role in different physiological processes. ROS scavenging is responsible for regulation of reactive oxygen species generation. A researcher has proved that microRNAs regulate tumor radiation resistance. Ionizing radiation does not kill the cancer cells; rather, IR just slows down the signs and symptoms. Ionizing radiation damages DNA directly/indirectly. IR is given mostly in combination with other chemo/radiotherapies. We briefly described here the behavior of cancer stem cells and radioresistance therapies in cancer treatment. To overcome radioresistance in treatment of cancer, strategies like fractionation modification, treatment in combination, inflammation modification, and overcoming hypoxic tumor have been practiced. Natural radiosensitizers, for example, curcumin, genistein, and quercetin, are more beneficial than synthetic compounds.

  4. Mosaic Analysis with Double Markers Reveals Cell-Type-Specific Paternal Growth Dominance

    Directory of Open Access Journals (Sweden)

    Simon Hippenmeyer

    2013-03-01

    Full Text Available Genomic imprinting leads to preferred expression of either the maternal or paternal alleles of a subset of genes. Imprinting is essential for mammalian development, and its deregulation causes many diseases. However, the functional relevance of imprinting at the cellular level is poorly understood for most imprinted genes. We used mosaic analysis with double markers (MADM in mice to create uniparental disomies (UPDs and to visualize imprinting effects with single-cell resolution. Although chromosome 12 UPD did not produce detectable phenotypes, chromosome 7 UPD caused highly significant paternal growth dominance in the liver and lung, but not in the brain or heart. A single gene on chromosome 7, encoding the secreted insulin-like growth factor 2 (IGF2, accounts for most of the paternal dominance effect. Mosaic analyses implied additional imprinted loci on chromosome 7 acting cell autonomously to transmit the IGF2 signal. Our study reveals chromosome- and cell-type specificity of genomic imprinting effects.

  5. Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells.

    Science.gov (United States)

    Nguyen, Long V; Pellacani, Davide; Lefort, Sylvain; Kannan, Nagarajan; Osako, Tomo; Makarem, Maisam; Cox, Claire L; Kennedy, William; Beer, Philip; Carles, Annaick; Moksa, Michelle; Bilenky, Misha; Balani, Sneha; Babovic, Sonja; Sun, Ivan; Rosin, Miriam; Aparicio, Samuel; Hirst, Martin; Eaves, Connie J

    2015-12-10

    Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop. PMID:26633636

  6. 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.

  7. Direct and Indirect Role of Toll-Like Receptors in T Cell Mediated Immunity

    Institute of Scientific and Technical Information of China (English)

    Damo Xu; Haiying Liu; Mousa Komai-Koma

    2004-01-01

    Toll-like receptors (TLR) are pathogen-associated molecular patterns (PAMPs) recognition receptors that play an important role in protective immunity against infection and inflammation. They act as central integrators of a wide variety of signals, responding to diverse agonists of microbial products. Stimulation of Toll-like receptors by microbial products leads to signaling pathways that activate not only innate, but also adaptive immunity by APC dependent or independent mechanisms. Recent evidence revealed that TLR signals played a determining role in the skewing of na(i)ve T cells towards either Th1 or Th2 responses. Activation of Toll-like receptors also directly or indirectly influences regulatory T cell functions. Therefore, TLRs are required in both immune activation and immune regulation. Study of TLRs has significantly enhanced our understanding of innate and adaptive immune responses and provides novel therapeutic approaches against infectious and inflammatory diseases.

  8. Direct and Indirect Role of Toll-Like Receptors in T Cell Mediated Immunity

    Institute of Scientific and Technical Information of China (English)

    DamoXu; HaiyingLiu; MousaKomai-Koma

    2004-01-01

    Toll-like receptors (TLR) are pathogen-associated molecular patterns (PAMPs) recognition receptors that play an important role in protective immunity against infection and inflammation. They act as central integrators of a wide variety of signals, responding to diverse agonists of microbial products. Stimulation of Toll-like receptors by microbial products leads to signaling pathways that activate not only innate, but also adaptive immunity by APC dependent or independent mechanisms. Recent evidence revealed that TLR signals played a determining role in the skewing of naive T cells towards either Thl or Th2 responses. Activation of Toll-like receptors also directly or indirectly influences regulatory T cell functions. Therefore, TLRs are required in both immune activation and immune regulation. Study of TLRs has significantly enhanced our understanding of innate and adaptive immune responses and provides novel therapeutic approaches against infectious and inflammatory diseases. Cellular & Molecular Immunology.

  9. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids...... to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present...... and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate...

  10. GSK-3: A Bifunctional Role in Cell Death Pathways

    Directory of Open Access Journals (Sweden)

    Keith M. Jacobs

    2012-01-01

    Full Text Available Although glycogen synthase kinase-3 beta (GSK-3β was originally named for its ability to phosphorylate glycogen synthase and regulate glucose metabolism, this multifunctional kinase is presently known to be a key regulator of a wide range of cellular functions. GSK-3β is involved in modulating a variety of functions including cell signaling, growth metabolism, and various transcription factors that determine the survival or death of the organism. Secondary to the role of GSK-3β in various diseases including Alzheimer’s disease, inflammation, diabetes, and cancer, small molecule inhibitors of GSK-3β are gaining significant attention. This paper is primarily focused on addressing the bifunctional or conflicting roles of GSK-3β in both the promotion of cell survival and of apoptosis. GSK-3β has emerged as an important molecular target for drug development.

  11. GSK-3β: A Bifunctional Role in Cell Death Pathways

    Science.gov (United States)

    Jacobs, Keith M.; Bhave, Sandeep R.; Ferraro, Daniel J.; Jaboin, Jerry J.; Hallahan, Dennis E.; Thotala, Dinesh

    2012-01-01

    Although glycogen synthase kinase-3 beta (GSK-3β) was originally named for its ability to phosphorylate glycogen synthase and regulate glucose metabolism, this multifunctional kinase is presently known to be a key regulator of a wide range of cellular functions. GSK-3β is involved in modulating a variety of functions including cell signaling, growth metabolism, and various transcription factors that determine the survival or death of the organism. Secondary to the role of GSK-3β in various diseases including Alzheimer's disease, inflammation, diabetes, and cancer, small molecule inhibitors of GSK-3β are gaining significant attention. This paper is primarily focused on addressing the bifunctional or conflicting roles of GSK-3β in both the promotion of cell survival and of apoptosis. GSK-3β has emerged as an important molecular target for drug development. PMID:22675363

  12. Integrative Analysis of the Acquisition of Pluripotency in PGCs Reveals the Mutually Exclusive Roles of Blimp-1 and AKT Signaling

    Directory of Open Access Journals (Sweden)

    Go Nagamatsu

    2015-07-01

    Full Text Available Primordial germ cells (PGCs are lineage-restricted unipotent cells that can dedifferentiate into pluripotent embryonic germ cells (EGCs. Here we performed whole-transcriptome analysis during the conversion of PGCs into EGCs, a process by which cells acquire pluripotency. To examine the molecular mechanism underlying this conversion, we focused on Blimp-1 and Akt, which are involved in PGC specification and dedifferentiation, respectively. Blimp-1 overexpression in embryonic stem cells suppressed the expression of downstream targets of the pluripotency network. Conversely, Blimp-1 deletion in PGCs accelerated their dedifferentiation into pluripotent EGCs, illustrating that Blimp-1 is a pluripotency gatekeeper protein in PGCs. AKT signaling showed a synergistic effect with basic fibroblast growth factor plus 2i+A83 treatment on EGC formation. AKT played a major role in suppressing genes regulated by MBD3. From these results, we defined the distinct functions of Blimp-1 and Akt and provided mechanistic insights into the acquisition of pluripotency in PGCs.

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

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    Mehdi Toufeer

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

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

    Science.gov (United States)

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

    2011-01-01

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

  15. Role of NK, NKT cells and macrophages in liver transplantation

    Science.gov (United States)

    Fahrner, René; Dondorf, Felix; Ardelt, Michael; Settmacher, Utz; Rauchfuss, Falk

    2016-01-01

    Liver transplantation has become the treatment of choice for acute or chronic liver disease. Because the liver acts as an innate immunity-dominant organ, there are immunological differences between the liver and other organs. The specific features of hepatic natural killer (NK), NKT and Kupffer cells and their role in the mechanism of liver transplant rejection, tolerance and hepatic ischemia-reperfusion injury are discussed in this review. PMID:27468206

  16. Subsets of regulatory T cells and their roles in allergy

    OpenAIRE

    ZHANG, HUIYUN; Kong, Hui; Zeng, Xiaoning; Guo, Lianyi; Sun, Xiaoyun; He, Shaoheng

    2014-01-01

    In recent years, it is recognized that acquired immunity is controlled by regulatory T cell (Treg). Since fundamental pathophysiological changes of allergy are mainly caused by hyperresponsiveness of immune system to allergens that acquires after birth, Tregs likely play key roles in the pathogenesis of allergy, particularly during the sensitization phase. However, accumulated information indicate that there are several distinctive subtypes of Tregs in man, and each of them seems to play diff...

  17. The role of regulatory T cells in cancer immunology

    OpenAIRE

    Whiteside TL

    2015-01-01

    Theresa L Whiteside University of Pittsburgh Cancer Institute, Pittsburgh, PA, US Abstract: Regulatory T cells (Treg) are generally considered to be significant contributors to tumor escape from the host immune system. Emerging evidence suggests, however, that in some human cancers, Treg are necessary to control chronic inflammation, prevent tissue damage, and limit inflammation-associated cancer development. The dual role of Treg in cancer and underpinnings of Treg diversity are not well und...

  18. Sterile inflammation - do innate lymphoid cell subsets play a role?

    OpenAIRE

    Walsh, Patrick

    2012-01-01

    PUBLISHED The recent identification of several novel innate lymphoid cell (iLC) subsets has increased our understanding of the mechanisms which link the innate and adaptive immune systems. While the contribution of these subsets toward the pathogenesis of human disease remains largely to be determined, it seems likely that they will play a particularly important role in sterile inflammatory settings where the innate response is seen as a critical mediator of inflammation. Several recent st...

  19. Transcriptome meta-analysis reveals a dysregulation in extra cellular matrix and cell junction associated gene signatures during Dengue virus infection

    Science.gov (United States)

    Afroz, Sumbul; Giddaluru, Jeevan; Abbas, Mohd. Manzar; Khan, Nooruddin

    2016-01-01

    Dengue Viruses (DENVs) cause one of the most prevalent arthropod-borne viral diseases affecting millions of people worldwide. Identification of genes involved in DENV pathogenesis would help in deciphering molecular mechanisms responsible for the disease progression. Here, we carried out a meta-analysis of publicly available gene expression data of dengue patients and further validated the meta-profile using in-vitro infection in THP-1 cells. Our findings reveal that DENV infection modulates expression of several genes and signalling pathways including interferons, detoxification of ROS and viral assembly. Interestingly, we have identified novel gene signatures comprising of INADL/PATJ and CRTAP (Cartilage Associated Protein), which were significantly down-regulated across all patient data sets as well as in DENV infected THP-1 cells. PATJ and CRTAP genes are involved in maintaining cell junction integrity and collagen assembly (extracellular matrix component) respectively, which together play a crucial role in cell-cell adhesion. Our results categorically reveal that overexpression of CRTAP and PATJ genes restrict DENV infection, thereby suggesting a critical role of these genes in DENV pathogenesis. Conclusively, these findings emphasize the utility of meta-analysis approach in identifying novel gene signatures that might provide mechanistic insights into disease pathogenesis and possibly lead towards the development of better therapeutic interventions. PMID:27651116

  20. Transcriptome meta-analysis reveals a dysregulation in extra cellular matrix and cell junction associated gene signatures during Dengue virus infection.

    Science.gov (United States)

    Afroz, Sumbul; Giddaluru, Jeevan; Abbas, Mohd Manzar; Khan, Nooruddin

    2016-01-01

    Dengue Viruses (DENVs) cause one of the most prevalent arthropod-borne viral diseases affecting millions of people worldwide. Identification of genes involved in DENV pathogenesis would help in deciphering molecular mechanisms responsible for the disease progression. Here, we carried out a meta-analysis of publicly available gene expression data of dengue patients and further validated the meta-profile using in-vitro infection in THP-1 cells. Our findings reveal that DENV infection modulates expression of several genes and signalling pathways including interferons, detoxification of ROS and viral assembly. Interestingly, we have identified novel gene signatures comprising of INADL/PATJ and CRTAP (Cartilage Associated Protein), which were significantly down-regulated across all patient data sets as well as in DENV infected THP-1 cells. PATJ and CRTAP genes are involved in maintaining cell junction integrity and collagen assembly (extracellular matrix component) respectively, which together play a crucial role in cell-cell adhesion. Our results categorically reveal that overexpression of CRTAP and PATJ genes restrict DENV infection, thereby suggesting a critical role of these genes in DENV pathogenesis. Conclusively, these findings emphasize the utility of meta-analysis approach in identifying novel gene signatures that might provide mechanistic insights into disease pathogenesis and possibly lead towards the development of better therapeutic interventions. PMID:27651116

  1. Mutations in Traf3ip1 reveal defects in ciliogenesis, embryonic development, and altered cell size regulation.

    Science.gov (United States)

    Berbari, Nicolas F; Kin, Nicholas W; Sharma, Neeraj; Michaud, Edward J; Kesterson, Robert A; Yoder, Bradley K

    2011-12-01

    Tumor necrosis factor alpha receptor 3 interacting protein 1 (Traf3ip1), also known as MIPT3, was initially characterized through its interactions with tubulin, actin, TNFR-associated factor-3 (Traf3), IL-13R1, and DISC1. It functions as an inhibitor of IL-13-mediated phosphorylation of Stat6 and in sequestration of Traf3 and DISC1 to the cytoskeleton. Studies of the Traf3ip1 homologs in C. elegans (DYF-11), Zebrafish (elipsa), and Chlamydomonas (IFT54) revealed that the protein localizes to the cilium and is required for ciliogenesis. Similar localization data has now been reported for mammalian Traf3ip1. This raises the possibility that Traf3ip1 has an evolutionarily conserved role in mammalian ciliogenesis in addition to its previously indicated functions. To evaluate this possibility, a Traf3ip1 mutant mouse line was generated. Traf3ip1 mutant cells are unable to form cilia. Homozygous Traf3ip1 mutant mice are not viable and have both neural developmental defects and polydactyly, phenotypes typical of mouse mutants with ciliary assembly defects. Furthermore, in Traf3ip1 mutants the hedgehog pathway is disrupted, as evidenced by abnormal dorsal-ventral neural tube patterning and diminished expression of a hedgehog reporter. Analysis of the canonical Wnt pathway indicates that it was largely unaffected; however, specific domains in the pharyngeal arches have elevated levels of reporter activity. Interestingly, Traf3ip1 mutant embryos and cells failed to show alterations in IL-13 signaling, one of the pathways associated with its initial discovery. Novel phenotypes observed in Traf3ip1 mutant cells include elevated cytosolic levels of acetylated microtubules and a marked increase in cell size in culture. The enlarged Traf3ip1 mutant cell size was associated with elevated basal mTor pathway activity. Taken together, these data demonstrate that Traf3ip1 function is highly conserved in ciliogenesis and is important for proper regulation of a number of essential

  2. 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.

  3. Role of Geminin in cell fate determination of hematopoietic stem cells (HSCs).

    Science.gov (United States)

    Yasunaga, Shin'ichiro; Ohno, Yoshinori; Shirasu, Naoto; Zhang, Bo; Suzuki-Takedachi, Kyoko; Ohtsubo, Motoaki; Takihara, Yoshihiro

    2016-09-01

    Geminin exerts two distinct molecular roles. Geminin negatively regulates DNA replication licensing through the direct interaction with Cdt1 to prevent re-replication in proliferating cells. Geminin also regulates chromatin remodeling through the direct interaction with Brahma/Brg1 to maintain undifferentiated states of stem cells. We previously uncovered that Polycomb-group complex 1 and Hoxb4/Hoxa9, well-known intrinsic factors that are essential for maintaining the hematopoietic stem cell (HSC) activity, alternatively act as ubiquitin-proteasome systems for Geminin protein to reduce the protein expression level, and sustain the HSC activity. Thus, Geminin is presumed to play an important role in determining cell fate, i.e., turning on and off cellular quiescence and proliferation/differentiation, in HSCs. We recently generated recombinant cell-penetrating Geminin (CP-Geminin), enabling rapid incorporation and withdraw of Geminin protein in cells. CP-Geminin may be useful in regulating the cell cycle and chromatin configuration. In this article, we summarize current information on the molecular functions of Geminin and the regulatory system for Geminin protein expression, and argue for the molecular role of Geminin in cell fate determination of HSCs, and future perspective of a new technology for manipulating the activities of HSCs and cancer stem cells (CSCs). PMID:27422432

  4. Interaction study of a novel Macrobrachium rosenbergii effector caspase with B2 and capsid proteins of M. rosenbergii nodavirus reveals their roles in apoptosis.

    Science.gov (United States)

    Youngcharoen, Supak; Senapin, Saengchan; Lertwimol, Tareerat; Longyant, Siwaporn; Sithigorngul, Paisarn; Flegel, Timothy W; Chaivisuthangkura, Parin

    2015-08-01

    Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of M. rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct protein-protein interaction between capsid protein and B2 protein of MrNV.

  5. Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging.

    Science.gov (United States)

    Kuznetsov, Andrey V; Hermann, Martin; Troppmair, Jakob; Margreiter, Raimund; Hengster, Paul

    2010-01-01

    Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission-fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1-0.3 mum/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 mum/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. PMID:19382913

  6. Research perspectives and role of lactose uptake rate revealed by its study using 14C-labelled lactose in whey fermentation.

    Science.gov (United States)

    Golfinopoulos, Aristidis; Kopsahelis, Nikolaos; Tsaousi, Konstantina; Koutinas, Athanasios A; Soupioni, Magdalini

    2011-03-01

    The present investigation examines the effect of pH, temperature and cell concentration on lactose uptake rate, in relation with kinetics of whey fermentation using kefir and determines the optimum conditions of these parameters. Lactose uptake rate was measured by adding (14)C-labelled lactose in whey. The results reveal the role of lactose uptake rate, being the main factor that affects the rate of fermentation, in contrast to the activity of the enzymes involved in lactose bioconversion process. Lactose uptake rate results discussion showed that mainly Ca(2+) is responsible for the reduced whey fermentation rate in comparison with fermentations using synthetic media containing lactose. Likewise, the results draw up perspectives on whey fermentation research to improve whey fermentation rate. Those perspectives are research to remove Ca(2+) from whey, the use of nano and microtubular biopolymers and promoters such as γ-alumina pellets and volcan foaming rock kissiris in order to accelerate whey fermentation. PMID:21232943

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

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

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

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

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    Siwo Geoffrey

    2010-10-01

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

  9. Biological role of NK cells and immunotherapeutic approaches in breast cancer

    Directory of Open Access Journals (Sweden)

    María Paula Roberti

    2012-12-01

    Full Text Available In recent decades, tumor surveillance by the immune system and its impact on disease outcomes in cancer patients in general and in breast cancer (BC patients in particular has been documented. Natural killer (NK cells are central components of the innate immunity and existing data indicate that they play a role in preventing and controlling tumor growth and metastasis. Their biological significance was first recognized by their ability to exert direct cellular cytotoxicity without prior sensitization. This is important in tumors, as transforming events are likely to result in downregulation of self-ligands and expression of stress-induced ligands which can be recognized by NK cells. Their activation also leads to secretion of stimulatory cytokines which participate in cancer elimination by several direct mechanisms as well as by stimulating the adaptive immune system. In this regard, it was recently revealed a dendritic cell (DC-NK cell crosstalk which provides another novel pathway linking innate and adaptive immunity. In addition, NK cells are feasible targets of stimulation in immunotherapeutic approaches such as antibody-based strategies and adoptive cell transfer. Nevertheless, NK cells display impaired functionality and capability to infiltrate tumors in BC patients. This review compiles information about NK cell biology in BC and the attempts which aim to manipulate them in novel therapeutic approaches in this pathology.

  10. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.

    Science.gov (United States)

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-08-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  11. Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection▿

    Science.gov (United States)

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B.; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L.; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-01-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  12. Identification of a negative regulatory role for spi-C in the murine B cell lineage.

    Science.gov (United States)

    Li, Stephen K H; Solomon, Lauren A; Fulkerson, Patricia C; DeKoter, Rodney P

    2015-04-15

    Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib(-/-)Spic(+/-)). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib(-/-) mouse spleens. Spib(-/-)Spic(+/-) B cells had restored proliferation compared with Spib(-/-) B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib(-/-)Spic(+/-) phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib(-/-)Spic(+/-) B cells compared with Spib(-/-) B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

  13. Identification of a negative regulatory role for spi-C in the murine B cell lineage.

    Science.gov (United States)

    Li, Stephen K H; Solomon, Lauren A; Fulkerson, Patricia C; DeKoter, Rodney P

    2015-04-15

    Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib(-/-)Spic(+/-)). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib(-/-) mouse spleens. Spib(-/-)Spic(+/-) B cells had restored proliferation compared with Spib(-/-) B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib(-/-)Spic(+/-) phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib(-/-)Spic(+/-) B cells compared with Spib(-/-) B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function. PMID:25769919

  14. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

    Science.gov (United States)

    Casero, David; Sandoval, Salemiz; Seet, Christopher S; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M

    2015-12-01

    To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

  15. Role of mucosal dendritic cells in inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Jan Hendrik Niess

    2008-01-01

    The gastrointestinal innate and adaptive immune system continuously faces the challenge of potent stimuli from the commensal microflora and food constituents.These local immune responses require a tight control,the outcome of which is in most cases the induction of tolerance.Local T cell immunity is an important compartment of the specific intestinal immune system.T cell reactivity is programmed during the initial stage of its activation by professional presenting cells.Mucosal dendritic cells(DCs)are assumed to play key roles in regulating immune responses in the antigen-rich gastrointestinal environment.Mucosal DCs are a heterogeneous population that can either initiate(innate and adaptive)immune responses,or control intestinal inflammation and maintain tolerance.Defects in this regulation are supposed to lead to the two major forms of inflammatory bowel disease(IBD),Crohn's disease(CD)and ulcerative colitis(UC).This review will discuss the emerging role of mucosal DCs in regulating intestinal inflammation and immune responses.(C)2008 The WJG Press.All rights reserved.

  16. Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

    Science.gov (United States)

    Mattei, Vincenzo; Santacroce, Costantino; Tasciotti, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Piccoli, Luca; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina

    2015-12-10

    Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-β-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation.

  17. The role of Cajal cells in chronic prostatitis.

    Science.gov (United States)

    Haki Yuksel, Ozgur; Urkmez, Ahmet; Verit, Ayhan

    2016-01-01

    Types of prostatitis can be defined as groups of syndromes in adult men associated with infectious and noninfectious causes characterized frequently by lower abdominal and perineal signs and diverse clinical symptoms and complications. Etiopathogenesis of chronic prostatitis is not well defined. Moreover, its treatment outcomes are not satisfactory. Presence of c-kit positive interstitial cells in human prostate is already known. It has been demonstrated that these cells can be pacemaker cells which trigger spontaneous slow-wave electrical activity in the prostate and can be responsible for the transport of glandular secretion from acinar cells into major and minor prostatic ducts and finally into urethra. In the light of all these data, when presence of a possible inflammatory pathology is thought to involve prostate that secretes and has a reservoir which drains its secretion (for prostate, prostatic urethra), two points are worth mentioning. Impairment of secretion mechanism and collection of secretion within the organ with reflux of the microbial material from its reservoir back into prostate gland. Both of these potential conditions can be explained by ductal neuromuscular mechanism, which induces secretion. We think that in this neuromuscular mechanism interstitial Cajal cells have an important role in chronic prostatitis. Our hypothesis is that curability of prostatitis is correlated with the number of Cajal cells not subjected to apoptosis. PMID:27377090

  18. The role of mast cells in vascularized recurrent pterygium

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    Ali Riza Cenk Celebi

    2014-10-01

    Full Text Available Objective: To determine and compare the mast cell count in primary and recurrent vascularized pterygium, and in normal bulbar conjunctiva. Methods: The study included 22 patients with primary pterygium (PP group and 28 patients with vascularized recurrent pterygium (VRP group that underwent excision via the limbal conjunctival autograft technique. Normal conjunctiva samples were collected from the superotemporal bulbar conjunctival region, just temporal to the site from which the autograft conjunctival tissue was harvested. The total number of mast cells in the pterygium (primary and recurrent and control tissue samples was calculated microscopically using 1% toluidine blue stain under 400× magnification. Results: The mean mast cell count in primary and vascularized recurrent pterygium tissue was 7.45 ± 2.06 mm–2 and 16.11 ± 4.33 mm–2, respectively, and the difference was significant (independent samples t-test, P<0.001. The mean mast cell count in pterygium tissue was significantly higher than that in normal conjunctiva tissue in both groups (Student's t-test, P<0.001. Conclusion: An increase in the number of mast cells might play a role in the pathogenesis of recurrent pterygium. Determination of a mast cell count cut-off value could be of diagnostic significance for recurrent pterygium.

  19. The Role of Lipid Domains in Bacterial Cell Processes

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    Katarína Muchová

    2013-02-01

    Full Text Available Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

  20. Cardiac stem cells and their roles in myocardial infarction.

    Science.gov (United States)

    Hou, Jingying; Wang, Lingyun; Jiang, Jieyu; Zhou, Changqing; Guo, Tianzhu; Zheng, Shaoxin; Wang, Tong

    2013-06-01

    Myocardial infarction leads to loss of cardiomyocytes, scar formation, ventricular remodeling and eventually deterioration of heart function. Over the past decade, stem cell therapy has emerged as a novel strategy for patients with ischemic heart disease and its beneficial effects have been demonstrated by substantial preclinical and clinical studies. Efficacy of several types of stem cells in the therapy of cardiovascular diseases has already been evaluated. However, repair of injured myocardium through stem cell transplantation is restricted by critical safety issues and ethic concerns. Recently, the discovery of cardiac stem cells (CSCs) that reside in the heart itself brings new prospects for myocardial regeneration and reconstitution of cardiac tissues. CSCs are positive for various stem cell markers and have the potential of self-renewal and multilineage differentiation. They play a pivotal role in the maintenance of heart homeostasis and cardiac repair. Elucidation of their biological characteristics and functions they exert in myocardial infarction are very crucial to further investigations on them. This review will focus on the field of cardiac stem cells and discuss technical and practical issues that may involve in their clinical applications in myocardial infarction.

  1. The role of B cells and autoantibodies in neuropsychiatric lupus.

    Science.gov (United States)

    Wen, Jing; Stock, Ariel D; Chalmers, Samantha A; Putterman, Chaim

    2016-09-01

    The central nervous system manifestations of SLE (neuropsychiatric lupus, NPSLE) occur frequently, though are often difficult to diagnose and treat. Symptoms of NPSLE can be quite diverse, including chronic cognitive and emotional manifestations, as well as acute presentations, such as stroke and seizures. Although the pathogenesis of NPSLE has yet to be well characterized, B-cell mediated damage is believed to be an important contributor. B-cells and autoantibodies may traverse the blood brain barrier promoting an inflammatory environment consisting of glia activation, neurodegeneration, and consequent averse behavioral outcomes. This review will evaluate the various suggested roles of B-cells and autoantibodies in NPSLE, as well as therapeutic modalities targeting these pathogenic mediators.

  2. Granulosa cell proliferation differentiation and its role in follicular development

    Institute of Scientific and Technical Information of China (English)

    LU Cuiling; YANG Wei; HU Zhaoyuan; LIU Yixun

    2005-01-01

    Granuiosa cells (GCs) are the most important cells in the ovary that undergo serious changes morphologically and physiologically during the processes of follicular proliferation, differentiation, ovulation, lutenization and atresia. Oocyte (OC) directs GC proliferation and differentiation, while GCs influence OC maturation. Many ovarian factors are involved in the regulation of these processes via different molecular mechanisms and signal pathways. P38MAPK can selectively regulate steroidogenesis in GCs controlled by FSH; Transcript factors LRH-1 and DAX-1 play an important role in this process; FSH induces GC prolfferation and differentiation by stimulating PCNA and StAR expression and steroidogenesis. Activated ERK1/2 signal pathway may be involved in the FSH-regulated GC proliferation and differentiation. Therefore, GC is an ideal model for studying cell proliferation, differentiation and interaction,as well as signal transduction. This review briefly summarizes the latest data in the literature, including the results achieved in our laboratory.

  3. Role And Relevance Of Mast Cells In Fungal Infections

    Directory of Open Access Journals (Sweden)

    Rohit eSaluja

    2012-06-01

    Full Text Available In addition to their detrimental role in allergic diseases, mast cells (MCs are well known to be important cells of the innate immune system. In the last decade, they have been shown to contribute significantly to optimal host defense against numerous pathogens including parasites, bacteria, and viruses. The contribution of MCs to the immune responses in fungal infections, however, is largely unknown. In this review, we first discuss key features of mast cell responses to pathogens in general and then summarize the current knowledge on the function of MCs in the defense against fungal pathogens. We especially focus on the potential and proven mechanisms by which MC can detect fungal infections and on possible MC effector mechanisms in protecting from fungal infections.

  4. Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

    Directory of Open Access Journals (Sweden)

    Chia Yin Lee

    2011-12-01

    Full Text Available Chikungunya virus (CHIKV is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

  5. Proteomic analysis of the mature Brassica stigma reveals proteins with diverse roles in vegetative and reproductive development.

    Science.gov (United States)

    Nazemof, Nazila; Couroux, Philippe; Xing, Tim; Robert, Laurian S

    2016-09-01

    The stigma, the specialized apex of the Brassicaceae gynoecium, plays a role in pollen capture, discrimination, hydration, germination, and guidance. Despite this crucial role in reproduction, the global proteome underlying Brassicaceae stigma development and function remains largely unknown. As a contribution towards the characterization of the Brassicaceae dry stigma global proteome, more than 2500 Brassica napus mature stigma proteins were identified using three different gel-based proteomics approaches. Most stigma proteins participated in Metabolic Processes, Responses to Stimulus or Stress, Cellular or Developmental Processes, and Transport. The stigma was found to express a wide variety of proteins with demonstrated roles in cellular and organ development including proteins known to be involved in cellular expansion and morphogenesis, embryo development, as well as gynoecium and stigma development. Comparisons to a corresponding proteome from a very morphologically different Poaceae dry stigma showed a very similar distribution of proteins among different functional categories, but also revealed evident distinctions in protein composition especially in glucosinolate and carotenoid metabolism, photosynthesis, and self-incompatibility. To our knowledge, this study reports the largest Brassicaceae stigma protein dataset described to date. PMID:27457983

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

    Science.gov (United States)

    Lanoiselée, Yann; Grebenkov, Denis S

    2016-05-01

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

  7. Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: Regulatory roles of cell surface glycans

    OpenAIRE

    Suzuki, Osamu; Abe, Masafumi

    2014-01-01

    Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic ac...

  8. Critical role of dendritic cells in T cell retention in the interfollicular region of Peyer's patches.

    Science.gov (United States)

    Obata, Takashi; Shibata, Naoko; Goto, Yoshiyuki; Ishikawa, Izumi; Sato, Shintaro; Kunisawa, Jun; Kiyono, Hiroshi

    2013-07-15

    Peyer's patches (PPs) simultaneously initiate active and quiescent immune responses in the gut. The immunological function is achieved by the rigid regulation of cell distribution and trafficking, but how the cell distribution is maintained remains to be elucidated. In this study, we show that binding of stromal cell-derived lymphoid chemokines to conventional dendritic cells (cDCs) is essential for the retention of naive CD4(+) T cells in the interfollicular region (IFR) of PPs. Transitory depletion of CD11c(high) cDCs in mice rapidly impaired the IFR structure in the PPs without affecting B cell follicles or germinal centers, lymphoid chemokine production from stromal cells, or the immigration of naive T cells into the IFRs of PPs. The cDC-orchestrated retention of naive T cells was mediated by heparinase-sensitive molecules that were expressed on cDCs and bound the lymphoid chemokine CCL21 produced from stromal cells. These data collectively reveal that interactions among cDCs, stromal cells, and naive T cells are necessary for the formation of IFRs in the PPs.

  9. Whole-brain circuit dissection in free-moving animals reveals cell-specific mesocorticolimbic networks

    Science.gov (United States)

    Michaelides, Michael; Anderson, Sarah Ann R.; Ananth, Mala; Smirnov, Denis; Thanos, Panayotis K.; Neumaier, John F.; Wang, Gene-Jack; Volkow, Nora D.; Hurd, Yasmin L.

    2013-01-01

    The ability to map the functional connectivity of discrete cell types in the intact mammalian brain during behavior is crucial for advancing our understanding of brain function in normal and disease states. We combined designer receptor exclusively activated by designer drug (DREADD) technology and behavioral imaging with μPET and [18F]fluorodeoxyglucose (FDG) to generate whole-brain metabolic maps of cell-specific functional circuits during the awake, freely moving state. We have termed this approach DREADD-assisted metabolic mapping (DREAMM) and documented its ability in rats to map whole-brain functional anatomy. We applied this strategy to evaluating changes in the brain associated with inhibition of prodynorphin-expressing (Pdyn-expressing) and of proenkephalin-expressing (Penk-expressing) medium spiny neurons (MSNs) of the nucleus accumbens shell (NAcSh), which have been implicated in neuropsychiatric disorders. DREAMM revealed discrete behavioral manifestations and concurrent engagement of distinct corticolimbic networks associated with dysregulation of Pdyn and Penk in MSNs of the NAcSh. Furthermore, distinct neuronal networks were recruited in awake versus anesthetized conditions. These data demonstrate that DREAMM is a highly sensitive, molecular, high-resolution quantitative imaging approach. PMID:24231358

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

    Science.gov (United States)

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

    2016-03-01

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

  11. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases.

    Science.gov (United States)

    Rokicki, Wojciech; Rokicki, Marek; Wojtacha, Jacek; Dżeljijli, Agata

    2016-03-01

    The report presents the cellular structure of the respiratory system as well as the history of club cells (Clara cells), their ultrastructure, and location in the airways and human organs. The authors discuss the biochemical structure of proteins secreted by these cells and their importance for the integrity and regeneration of the airway epithelium. Their role as progenitor cells for the airway epithelium and their involvement in the biotransformation of toxic xenobiotics introduced into the lungs during breathing is emphasized. This is followed by a discussion of the clinical aspects associated with club cells, demonstrating that tracking the serum concentration of club cell-secreted proteins is helpful in the diagnosis of a number of lung tissue diseases. Finally, suggestions are provided regarding the possible use of proteins secreted by club cells in the treatment of serious respiratory conditions. PMID:27212975

  12. High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker

    Science.gov (United States)

    Choi, He Yun; Park, Ji Hye; Jang, Woong Bi; Ji, Seung Taek; Jung, Seok Yun; Kim, Da Yeon; Kang, Songhwa; Kim, Yeon Ju; Yun, Jisoo; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang-Mo

    2016-01-01

    Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes. PMID:27350339

  13. A cell cycle role for the epigenetic factor CTCF-L/BORIS.

    Directory of Open Access Journals (Sweden)

    Manuel Rosa-Garrido

    Full Text Available CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

  14. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.

    Directory of Open Access Journals (Sweden)

    Mona Saffarzadeh

    Full Text Available Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET. These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

  15. Cell model of catecholaminergic polymorphic ventricular tachycardia reveals early and delayed afterdepolarizations.

    Directory of Open Access Journals (Sweden)

    Kirsi Kujala

    Full Text Available BACKGROUND: Induced pluripotent stem cells (iPSC provide means to study the pathophysiology of genetic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT is a malignant inherited ion channel disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2. In this study the cellular characteristics of CPVT are investigated and whether the electrophysiological features of this mutation can be mimicked using iPSC -derived cardiomyocytes (CM. METHODOLOGY/PRINCIPAL FINDINGS: Spontaneously beating CMs were differentiated from iPSCs derived from a CPVT patient carrying a P2328S mutation in RyR2 and from two healthy controls. Calcium (Ca(2+ cycling and electrophysiological properties were studied by Ca(2+ imaging and patch-clamp techniques. Monophasic action potential (MAP recordings and 24h-ECGs of CPVT-P2328S patients were analyzed for the presence of afterdepolarizations. We found defects in Ca(2+ cycling and electrophysiology in CPVT CMs, reflecting the cardiac phenotype observed in the patients. Catecholaminergic stress led to abnormal Ca(2+ signaling and induced arrhythmias in CPVT CMs. CPVT CMs also displayed reduced sarcoplasmic reticulum (SR Ca(2+ content, indicating leakage of Ca(2+ from the SR. Patch-clamp recordings of CPVT CMs revealed both delayed afterdepolarizations (DADs during spontaneous beating and in response to adrenaline and also early afterdepolarizations (EADs during spontaneous beating, recapitulating the changes seen in MAP and 24h-ECG recordings of patients carrying the same mutation. CONCLUSIONS/SIGNIFICANCE: This cell model shows aberrant Ca(2+ cycling characteristic of CPVT and in addition to DADs it displays EADs. This cell model for CPVT provides a platform to study basic pathology, to screen drugs, and to optimize drug therapy.

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

    Science.gov (United States)

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

    2016-05-01

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

  17. Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

    Science.gov (United States)

    Narushima, Yuta; Kozuka-Hata, Hiroko; Koyama-Nasu, Ryo; Tsumoto, Kouhei; Inoue, Jun-ichiro; Akiyama, Tetsu; Oyama, Masaaki

    2016-03-01

    Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation. PMID:26670566

  18. Role of endodermal cell vacuoles in shoot gravitropism.

    Science.gov (United States)

    Kato, Takehide; Morita, Miyo Terao; Tasaka, Masao

    2002-06-01

    In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.

  19. The Role of the Immune Response in Merkel Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Triozzi, Pierre L., E-mail: triozzp@ccf.org [Taussig Cancer Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Fernandez, Anthony P. [Departments of Dermatology and Anatomic Pathology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 (United States)

    2013-02-28

    Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer. The Merkel cell polyomavirus (MCPyV) is implicated in its pathogenesis. Immune mechanisms are also implicated. Patients who are immunosuppressed have an increased risk. There is evidence that high intratumoral T-cell counts and immune transcripts are associated with favorable survival. Spontaneous regressions implicate immune effector mechanisms. Immunogenicity is also supported by observation of autoimmune paraneoplastic syndromes. Case reports suggest that immune modulation, including reduction of immune suppression, can result in tumor regression. The relationships between MCPyV infection, the immune response, and clinical outcome, however, remain poorly understood. Circulating antibodies against MCPyV antigens are present in most individuals. MCPyV-reactive T cells have been detected in both MCC patients and control subjects. High intratumoral T-cell counts are also associated with favorable survival in MCPyV-negative MCC. That the immune system plays a central role in preventing and controlling MCC is supported by several observations. MCCs often develop, however, despite the presence of humoral and cellular immune responses. A better understanding on how MCPyV and MCC evade the immune response will be necessary to develop effective immunotherapies.

  20. Role of mitochondrial function in cell death and body metabolism.

    Science.gov (United States)

    Lee, Myung-Shik

    2016-01-01

    Mitochondria are the key players in apoptosis and necrosis. Mitochondrial DNA (mtDNA)-depleted r0 cells were resistant to diverse apoptosis inducers such as TNF-alpha, TNFSF10, staurosporine and p53. Apoptosis resistance was accompanied by the absence of mitochondrial potential loss or cytochrome c translocation. r0 cells were also resistant to necrosis induced by reactive oxygen species (ROS) donors due to upregulation of antioxidant enzymes such as manganese superoxide dismutase. Mitochondria also has a close relationship with autophagy that plays a critical role in the turnover of senescent organelles or dysfunctional proteins and may be included in 'cell death' category. It was demonstrated that autophagy deficiency in insulin target tissues such as skeletal muscle induces mitochondrial stress response, which leads to the induction of FGF21 as a 'mitokine' and affects the whole body metabolism. These results show that mitochondria are not simply the power plants of cells generating ATP, but are closely related to several types of cell death and autophagy. Mitochondria affect various pathophysiological events related to diverse disorders such as cancer, metabolic disorders and aging. PMID:27100503

  1. Deep sequencing of virus-infected cells reveals HIV-encoded small RNAs.

    Science.gov (United States)

    Schopman, Nick C T; Willemsen, Marcel; Liu, Ying Poi; Bradley, Ted; van Kampen, Antoine; Baas, Frank; Berkhout, Ben; Haasnoot, Joost

    2012-01-01

    Small virus-derived interfering RNAs (viRNAs) play an important role in antiviral defence in plants, insects and nematodes by triggering the RNA interference (RNAi) pathway. The role of RNAi as an antiviral defence mechanism in mammalian cells has been obscure due to the lack of viRNA detection. Although viRNAs from different mammalian viruses have recently been identified, their functions and possible impact on viral replication remain unknown. To identify viRNAs derived from HIV-1, we used the extremely sensitive SOLiD(TM) 3 Plus System to analyse viRNA accumulation in HIV-1-infected T lymphocytes. We detected numerous small RNAs that correspond to the HIV-1 RNA genome. The majority of these sequences have a positive polarity (98.1%) and could be derived from miRNAs encoded by structured segments of the HIV-1 RNA genome (vmiRNAs). A small portion of the viRNAs is of negative polarity and most of them are encoded within the 3'-UTR, which may represent viral siRNAs (vsiRNAs). The identified vsiRNAs can potently repress HIV-1 production, whereas suppression of the vsiRNAs by antagomirs stimulate virus production. These results suggest that HIV-1 triggers the production of vsiRNAs and vmiRNAs to modulate cellular and/or viral gene expression. PMID:21911362

  2. Haploinsufficiency after successive loss of signaling reveals a role for ERECTA-family genes in Arabidopsis ovule development.

    Science.gov (United States)

    Pillitteri, Lynn Jo; Bemis, Shannon M; Shpak, Elena D; Torii, Keiko U

    2007-09-01

    The Arabidopsis genome contains three ERECTA-family genes, ERECTA (ER), ERECTA-LIKE 1 (ERL1) and ERL2 that encode leucine-rich repeat receptor-like kinases. This gene family acts synergistically to coordinate cell proliferation and growth during above-ground organogenesis with the major player, ER, masking the loss-of-function phenotypes of the other two members. To uncover the specific developmental consequence and minimum threshold requirement for signaling, ER-family gene function was successively eliminated. We report here that ERL2 is haploinsufficient for maintaining female fertility in the absence of ER and ERL1. Ovules of the haploinsufficient er-105 erl1-2 erl2-1/+ mutant exhibit abnormal development with reduced cell proliferation in the integuments and gametophyte abortion. Our analysis indicates that progression of integument growth requires ER-family signaling in a dosage-dependent manner and that transcriptional compensation among ER-family members occurs to maintain the required signaling threshold. The specific misregulation of cyclin A genes in the er-105 erl1-2 erl2-1/+ mutant suggests that downstream targets of the ER-signaling pathway might include these core cell-cycle regulators. Finally, genetic interaction of the ER family and the WOX-family gene, PFS2, reveals their contribution to integument development through interrelated mechanisms. PMID:17652352

  3. Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle BiogenesisW⃞

    Science.gov (United States)

    Lurin, Claire; Andrés, Charles; Aubourg, Sébastien; Bellaoui, Mohammed; Bitton, Frédérique; Bruyère, Clémence; Caboche, Michel; Debast, Cédrig; Gualberto, José; Hoffmann, Beate; Lecharny, Alain; Le Ret, Monique; Martin-Magniette, Marie-Laure; Mireau, Hakim; Peeters, Nemo; Renou, Jean-Pierre; Szurek, Boris; Taconnat, Ludivine; Small, Ian

    2004-01-01

    The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms. PMID:15269332

  4. Revealing the role of oxidation state in interaction between nitro/amino-derived particulate matter and blood proteins

    Science.gov (United States)

    Liu, Zhen; Li, Ping; Bian, Weiwei; Yu, Jingkai; Zhan, Jinhua

    2016-05-01

    Surface oxidation states of ultrafine particulate matter can influence the proinflammatory responses and reactive oxygen species levels in tissue. Surface active species of vehicle-emission soot can serve as electron transfer-mediators in mitochondrion. Revealing the role of surface oxidation state in particles-proteins interaction will promote the understanding on metabolism and toxicity. Here, the surface oxidation state was modeled by nitro/amino ligands on nanoparticles, the interaction with blood proteins were evaluated by capillary electrophoresis quantitatively. The nitro shown larger affinity than amino. On the other hand, the affinity to hemoglobin is 103 times larger than that to BSA. Further, molecular docking indicated the difference of binding intensity were mainly determined by hydrophobic forces and hydrogen bonds. These will deepen the quantitative understanding of protein-nanoparticles interaction from the perspective of surface chemical state.

  5. The Role of Mast Cells in Alzheimer's Disease.

    Science.gov (United States)

    Shaik-Dasthagirisaheb, Yasdani B; Conti, Pio

    2016-01-01

    Immunity and inflammation are deeply involved in Alzheimer's disease. The most important properties of pathological Alzheimer's disease are the extracellular deposits of amyloid â-protein plaque aggregates along with other unknown mutated proteins, which are implicated in immunity and inflammation. Mast cells are found in the brain of all mammalian species and in the periphery, and their biological mediators, including cytokines/chemokines, arachidonic acid products and stored enzymes, play an import role in Alzheimer's disease. Cytokines/chemokines, which are generated mostly by microglia and astrocytes in Alzheimer's disease, contribute to nearly every aspect of neuroinflammation and amyloid â-protein plaque aggregates may induce in mast cells the release of a plethora of mediators, including pro-inflammatory cytokines/chemokines such as interleukin-1, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha, vascular endothelial growth factor, transforming growth factor beta, CXCL8 and CCL2-3-4. These proinflammatory cytokines/chemokines are prominent mediators of neuroinflammation in brain disorders such as Alzheimer's disease, and their inhibition may be associated with improved recovery. In this review, we summarize the current knowledge regarding the roles of mast cell mediators (stored and de novo synthesis) in the pathogenesis of Alzheimer's disease. PMID:27629855

  6. Regionally-specified second trimester fetal neural stem cells reveals differential neurogenic programming.

    Directory of Open Access Journals (Sweden)

    Yiping Fan

    Full Text Available Neural stem/progenitor cells (NSC have the potential for treatment of a wide range of neurological diseases such as Parkinson Disease and multiple sclerosis. Currently, NSC have been isolated only from hippocampus and subventricular zone (SVZ of the adult brain. It is not known whether NSC can be found in all parts of the developing mid-trimester central nervous system (CNS when the brain undergoes massive transformation and growth. Multipotent NSC from the mid-trimester cerebra, thalamus, SVZ, hippocampus, thalamus, cerebellum, brain stem and spinal cord can be derived and propagated as clonal neurospheres with increasing frequencies with increasing gestations. These NSC can undergo multi-lineage differentiation both in vitro and in vivo, and engraft in a developmental murine model. Regionally-derived NSC are phenotypically distinct, with hippocampal NSC having a significantly higher neurogenic potential (53.6% over other sources (range of 0%-27.5%, p<0.004. Whole genome expression analysis showed differential gene expression between these regionally-derived NSC, which involved the Notch, epidermal growth factor as well as interleukin pathways. We have shown the presence of phenotypically-distinct regionally-derived NSC from the mid-trimester CNS, which may reflect the ontological differences occurring within the CNS. Aside from informing on the role of such cells during fetal growth, they may be useful for different cellular therapy applications.

  7. Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells.

    Science.gov (United States)

    Singh, Jagdeep K; Simões, Bruno M; Howell, Sacha J; Farnie, Gillian; Clarke, Robert B

    2013-01-01

    Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.

  8. Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation.

    Science.gov (United States)

    Tokunaga, Hiroki; Kojima, Mikiko; Kuroha, Takeshi; Ishida, Takashi; Sugimoto, Keiko; Kiba, Takatoshi; Sakakibara, Hitoshi

    2012-01-01

    Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.

  9. Roles of Nrf2 in cell proliferation and differentiation.

    Science.gov (United States)

    Murakami, Shohei; Motohashi, Hozumi

    2015-11-01

    The Keap1-Nrf2 system plays pivotal roles in defense mechanisms by regulating cellular redox homeostasis. Nrf2 is an inducible transcription factor that activates a battery of genes encoding antioxidant proteins and phase II enzymes in response to oxidative stress and electrophilic xenobiotics. The activity of Nrf2 is regulated by Keap1, which promotes the ubiquitination and subsequent degradation of Nrf2 under normal conditions and releases the inhibited Nrf2 activity upon exposure to the stresses. Though an impressive contribution of the Keap1-Nrf2 system to the protection from exogenous and endogenous electrophilic insults has been well established, a line of evidence has suggested that the Keap1-Nrf2 system has various novel functions, particularly in cell proliferation and differentiation. Because the proliferation and differentiation of diverse cell types are often influenced and modulated by the cellular redox balance, Nrf2 has been considered to control these cellular processes by regulating the cellular levels of reactive oxygen species (ROS). In addition, analyses of the genome-wide distribution of Nrf2 have identified new sets of Nrf2 target genes whose products are involved in cell proliferation and differentiation but not necessarily in the regulation of oxidative stress. Considering the most characteristic features of Nrf2 as an inducible transcription factor, a newly emerged concept proposes that the Keap1-Nrf2 system translates environmental stresses into regulatory network signals in cell fate determination. In this review, we introduce the contribution of Nrf2 to lineage-specific differentiation, maintenance and differentiation of stem cells, and proliferation of normal and cancer cells, and we discuss how the response to fluctuating environments modulates cell behavior through the Keap1-Nrf2 system. PMID:26119783

  10. Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut.

    Directory of Open Access Journals (Sweden)

    Sudarshan Anand Shetty

    Full Text Available With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563 and BL7 (DSM25562, isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460 highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate, vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the

  11. Disruption of Arabidopsis CHY1 Reveals an Important Role of Metabolic Status in Plant Cold Stress Signaling

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Bethany K. Zolman; Bonnie Bartel; Byeong-ha Lee; Becky Stevenson; Manu Agarwal; Jian-Kang Zhu

    2009-01-01

    To study cold signaling, we screened for Arabidopsis mutants with altered cold-induced transcription of a firefly luciferase reporter gene driven by the CBF3 promoter (CBF3-LUC). One mutant, chyl-10, displayed reduced cold-induction of CBF3-LUC luminescence. RNA gel blot analysis revealed that expression of endogenous CBFs also was reduced in the chy1 mutant, chyl-10 mutant plants are more sensitive to freezing treatment than wild-type after cold acclimation. Both the wild-type and chy1 mutant plants are sensitive to darkness-induced starvation at warm temperatures, although chy1 plants are slightly more sensitive. This dark-sensitivity is suppressed by cold temperature in the wildtype but not in chy1. Constitutive CBF3 expression partially rescues the sensitivity of chy1-10 plants to dark treatment in the cold. The chy1 mutant accumulates higher levels of reactive oxygen species, and application of hydrogen peroxide can reduce cold-induction of CBF3-LUC in wild-type. Map-based cloning of the gene defective in the mutant revealed a nonsense mutation in CHY1, which encodes a peroxisomal β-hydroxyisobutyryl (HIBYL)-CoA hydrolase needed for valine catabolism and fatty acid β-oxidation. Our results suggest a role for peroxisomal metabolism in cold stress signaling, and plant tolerance to cold stress and darkness-induced starvation.

  12. Analysis of purified glabra3-shapeshifter trichomes reveals a role for NOECK in regulating early trichome morphogenic events.

    Science.gov (United States)

    Gilding, Edward K; Marks, M David

    2010-10-01

    Transcriptome analysis using the Affymetrix ATH1 platform has been completed on purified trichomes from the gl3-sst mutant. These trichomes display immature features, such as glassy cell walls and blunted branches. The gl3-sst trichome transcriptome was greatly enriched for genes involved in lipid biosynthesis, including those mediating the synthesis of fatty acids and wax. In addition, gl3-sst trichomes displayed reduced expression of the R3 MYBs TRY and CPC, which normally function to limit trichome development. The expression of the MIXTA-like MYB gene NOK was elevated. Members of the MIXTA-like family promote conical cell outgrowth, and in some cases, trichome initiation in diverse plant species. In contrast, NOK limits trichome outgrowth in wild-type Arabidopsis plants. Similar to other MIXTA-like genes, NOK was required for the expansion of gl3-sst trichomes, as the gl3-sst nok double mutant trichomes were greatly reduced in size. Expression of NOK in nok mutants reduced branch formation, whereas in gl3-sst nok, NOK expression promoted trichome cell outgrowth, illustrating duel roles for NOK in both promoting and limiting trichome development. MIXTA-like genes from phylogenetically diverse plant species could substitute for NOK in both nok and gl3-sst nok backgrounds. These findings suggest that certain aspects of NOK and MIXTA-like gene function have been conserved. PMID:21070410

  13. Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.

  14. Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

    Institute of Scientific and Technical Information of China (English)

    ZHANG TingTing; LI WanJie; LI Di; WANG Yue; SANG JianLi

    2008-01-01

    Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25△/△ mutants and investigated the role of the gene In morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25△/△ mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.

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

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

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