Science.gov

Sample records for 1-positive cells reveals

  1. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells.

    PubMed

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B; Bischoff, Joyce

    2015-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation, and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2, they converted to a mesenchymal phenotype after 3 weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to redifferentiate into endothelial cells, or into pericytes/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in IH.

  2. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells

    PubMed Central

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B.; Bischoff, Joyce

    2014-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin and VEGFR2, they converted to a mesenchymal phenotype after three weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to re-differentiate into endothelial cells, or into pericyte/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in infantile hemangioma. PMID:25187207

  3. Stem cell populations in the heart and the role of Isl1 positive cells.

    PubMed

    Di Felice, V; Zummo, G

    2013-05-09

    Cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells, endothelial cells and cardiomyocytes. The embryonic origin of these cells has not yet been demonstrated, but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest. In the last decade, two diffe-rent populations of cardiac progenitor or stem cells have been identified and isolated, i.e., the Islet1 positive (Isl1+) and c-Kit positive (c-Kit+)/Stem Cell Antigen-1 positive (Sca-1+) cells. Until 2012, these two populations have been considered two separate entities with different roles and a different origin, but new evidence now suggests a con-nection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells, derived from a common immature primitive cell. To find a common consensus on this concept is very important in furthe-ring the application of stem cells to cardiac tissue engineering.

  4. A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

    PubMed

    Turner, Elizabeth C; Huang, Chien-Ling; Sawhney, Neha; Govindarajan, Kalaimathi; Clover, Anthony J P; Martin, Kenneth; Browne, Tara C; Whelan, Derek; Kumar, Arun H S; Mackrill, John J; Wang, Shaohua; Schmeckpeper, Jeffrey; Stocca, Alessia; Pierce, William G; Leblond, Anne-Laure; Cai, Liquan; O'Sullivan, Donnchadh M; Buneker, Chirlei K; Choi, Janet; MacSharry, John; Ikeda, Yasuhiro; Russell, Stephen J; Caplice, Noel M

    2016-05-01

    Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC.  In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement.  PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

  5. Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

    PubMed

    Leppänen, Joni; Helminen, Olli; Huhta, Heikki; Kauppila, Joonas H; Miinalainen, Ilkka; Ronkainen, Veli-Pekka; Saarnio, Juha; Lehenkari, Petri P; Karttunen, Tuomo J

    2016-11-01

    Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

  6. Fibrocytes Regulate Wilms’ Tumor 1-Positive Cell Accumulation in Severe Fibrotic Lung Disease

    PubMed Central

    Sontake, Vishwaraj; Shanmukhappa, Shiva K.; DiPasquale, Betsy A.; Reddy, Geereddy B.; Medvedovic, Mario; Hardie, William D.; White, Eric S.; Madala, Satish K.

    2015-01-01

    Collagen-producing myofibroblast transdifferentiation is considered a crucial determinant in the formation of scar tissue in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Multiple resident pulmonary cell types and bone marrow-derived fibrocytes have been implicated as contributors to fibrotic lesions due to the transdifferentiation potential of these cells into myofibroblasts. In this study, we assessed the expression of Wilms’ tumor 1 (WT1), a known marker of mesothelial cells, in various cell types in normal and fibrotic lungs. We demonstrate that WT1 is expressed by both mesothelial and mesenchymal cells in IPF lungs, but has limited or no expression in normal human lungs. We also demonstrate that WT1-positive cells accumulate in fibrotic lung lesions, using two different mouse models of pulmonary fibrosis and WT1 promoter-driven fluorescent reporter mice. Reconstitution of bone-marrow cells into a transforming growth factor-α transgenic-mouse model demonstrated that fibrocytes do not transform into WT1-positive mesenchymal cells, but do augment accumulation of WT1-positive cells in severe fibrotic lung disease. Importantly, the number of WT1-positive cells in fibrotic lesions were correlated with severity of lung disease as assessed by changes in lung function, histology, and hydroxyproline levels in mice. Finally, inhibition of WT1 expression was sufficient to attenuate collagen and other extracellular-matrix gene production by mesenchymal cells from both murine and human fibrotic lungs. Thus, the results of this study demonstrate a novel association between fibrocyte-driven WT1-positive cell accumulation and severe fibrotic lung disease. PMID:26371248

  7. Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity.

    PubMed

    Rasper, Michael; Schäfer, Andrea; Piontek, Guido; Teufel, Julian; Brockhoff, Gero; Ringel, Florian; Heindl, Stefan; Zimmer, Claus; Schlegel, Jürgen

    2010-10-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs, we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently, the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated, stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast, ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results.

  8. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    PubMed Central

    Abouzaripour, Morteza; Pasbakhsh, Parichehr; Atlasi, Nader; Shahverdi, Abdol Hossein; Mahmoudi, Reza; Kashani, Iraj Ragerdi

    2016-01-01

    Objective Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion Our study clearly demonstrates the potential of SSEA-1 positive

  9. Complex chromosomal abnormalities in a patient with HTLV-1 positive T-cell leukemia

    SciTech Connect

    Hyde, P.; Macera, M.J.; Gogineni, S.K.

    1994-09-01

    HTLV-1 positive adult T-cell leukemia (ATL) is associated with numerous chromosomal abnormalities. The chromosomal rearrangements can be extremely complex and additional material is often present, making precise identification by routine cytogenetic techniques difficult. We report a case of ATL that was established of bone marrow cells by both QFQ and GTG banding techniques revealed a highly complex 49,XX,der(2)t(2;?)(q37;?),+5,+2mar karyotype in the dividing cells. The identical cytogenetic findings were also seen in unstimulated peripheral blood collected one week later. Using the FISH-technique, we applied spectrum green-labeled No. 1- and No. 7-specific WCP, spectrum orange-labeled No. 2- and No. 5-specific WCP (GIBCO/BRL, Gaithersburg, MD) and biotin-labeled No. 18-specific WCP (Oncor, Gaithersburg, MD) to metaphase chromosomes. The large marker chromosome was identified as an extra 1q arm, the material attached to the distal 2q was additional 7q. The presence of three No. 5 chromosomes was verified and the small marker was determined to be an extra partial 5p in Robertsonian translocation with an additional partial 18q arm. The karyotype was revised to 49,XX,+1q,der(2)t(2;7)(q37;q22),+5,+t(5;18)(p14{r_arrow}p11::q11{r_arrow}q12). Identification of the numerous chromosomal anomalies associated with the disease by molecular techniques shall lead to a better understanding of this deadly cancer.

  10. IFATS collection: Stem cell antigen-1-positive ear mesenchymal stem cells display enhanced adipogenic potential.

    PubMed

    Staszkiewicz, Jaroslaw; Gimble, Jeffrey M; Manuel, Jessica A; Gawronska-Kozak, Barbara

    2008-10-01

    Hyperplasia is a major contributor to the increase in adipose tissue mass that is characteristic of obesity. However, the identity and characteristics of cells that can be committed into adipocyte lineage remain unclear. Stem cell antigen 1 (Sca-1) has been used recently as a candidate marker in the search for tissue-resident stem cells. In our quest for biomarkers of cells that can become adipocytes, we analyzed ear mesenchymal stem cells (EMSC), which can differentiate into adipocytes, osteocytes, chondrocytes, and myocytes. Our previous studies have demonstrated that EMSC abundantly expressed Sca-1. In the present study, we have analyzed the expression of adipogenic transcription factors and adipocyte-specific genes in Sca-1-enriched and Sca-1-depleted EMSC fractions. Sca-1-enriched EMSC accumulated more lipid droplets during adipogenic differentiation than Sca-1-depleted. Similarly, EMSC isolated from Sca-1(-/-) mice displayed reduced lipid accumulation relative to EMSC from wild-type controls (p < .01). Comparative analysis of the adipogenic differentiation process between Sca-1-enriched and Sca-1-depleted populations of EMSC revealed substantial differences in the gene expression. Preadipocyte factor 1, CCAAT enhancer-binding protein (C/EBP) beta, C/EBPalpha, peroxisome proliferator-activated receptor gamma2, lipoprotein lipase, and adipocyte fatty acid binding protein were expressed at significantly higher levels in the Sca-1-enriched EMSC fraction. However, the most striking observation was that leptin was detected only in the conditioned medium of Sca-1-enriched EMSC. In addition, we performed loss-of-function (Sca-1 morpholino oligonucleotide) experiments. The data presented here suggest that Sca-1 is a biomarker for EMSC with the potential to become functionally active adipocytes. Disclosure of potential conflicts of interest is found at the end of this article.

  11. Antibody-directed double suicide gene therapy targeting of MUC1- positive leukemia cells in vitro and in vivo.

    PubMed

    Dong, Xiao-Ya; Wang, Wen-Qian; Zhao, Yu; Li, Xu-Dong; Fang, Zhi-Gang; Lin, Dong-Jun; Xiao, Ruo-Zhi; Huang, Ren-Wei; Pan, Guang-Jin; Liu, Jia-Jun

    2013-10-01

    Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

  12. Fisetin inhibits migration, invasion and epithelial-mesenchymal transition of LMP1-positive nasopharyngeal carcinoma cells.

    PubMed

    Li, Rong; Zhao, Yinhai; Chen, Jin; Shao, Songjun; Zhang, Xiujuan

    2014-02-01

    Fisetin (3,3',4',7-tetrahydroxyflavone) has been reported to possess certain anticancer properties. It may inhibit tumor cell proliferation, metastasis and induce apoptosis. However, the effects of fisetin in preventing the metastasis of nasopharyngeal carcinoma (NPC) cells remain to be determined. The epithelial-mesenchymal transition (EMT) is involved in several metastatic malignancies including NPC. It has been reported that the Epstein-Barr virus latent membrane protein-1 (LMP1) induced EMT and is associated with the metastasis of NPC. The aim of this study was to examine the effects of fisetin in preventing the migration and invasion of LMP1-expressing NPC cells (CNE1-LMP1 cells), as well as to investigate whether fisetin may inhibit the molecular changes associated with EMT induced by LMP1. The investigation demonstrated that fisetin suppressed the migration and invasion of CNE1-LMP1 cells under non-cytotoxic concentrations. Fisetin inhibited molecular changes associated with EMT induced by LMP1, upregulated the epithelial marker, E-cadherin protein, and downregulated the mesenchymal marker, vimentin protein, levels. Fisetin also significantly reduced the levels of Twist protein, an EMT regulator. The investigation suggested that fisetin inhibits the migration and invasion of LMP1-positive NPC cells, and the molecular mechanism involves fisetin reversing the EMT induced by LMP1 and downregulates the expression of Twist. This study indicated that fisetin serves as a potential candidate for the treatment of cancer metastasis.

  13. Conversion of Normal Ly-1-Positive B-Lineage Cells into Ly-1-Positive Macrophages in Long-Term Bone Marrow Cultures

    PubMed Central

    Katoh, Shigeki; Tominaga, Akira; Migita, Masahiro; Kudo, Akira

    1990-01-01

    We obtained eight different cell lines in the long-term bone marrow culture system that showed a germ-line configuration of the joining (J) region segments of the Ig heavy-chain (IgH) genes. Their surface markers were CD45R+, Ly-1+, Lyb-2+, cIgM-, sIgM-, Ia-, Thy-1-, Mac-1-, and IL-2R (Tac)+. Use of very young mice and the presence of IL-5 were important for preferential promotion of the survival of B-lineage lymphocytes bearing the Ly-1 markers. When we treated two of them (J8 and J10) with 5-azacytidine for 24 h followed by co-culture with stromal cells and IL-.5, they became Ly-1+, sIgM+ B cells, and Ly-1+, Mac-1+ macrophagelike cells, respectively. After other early lymphoid lines (J1, J8, and J13) were maintained by co-culture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene, although only J13 rearranged the κ-light chain gene. Northern blot analysis revealed that these cell lines expressed Cμ-mRNA, and λ5-mRNA, consistent with normal pre-B cells. Intriguingly, J1, J8, and J13 expressed c-fms mRNA constitutively. When J13 cells were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspecific-esterase-positive, and showed phagocytosis of latex beads. These results support evidence for a close relationship between the myeloid and Ly-1+ B-cell pathways of differentiation, and indicate that our IL- 5-dependent clones are multipotential intermediates in differentiation from pro-B cells to B cells and macrophages. PMID:2136207

  14. Bmi1-positive cells in the lingual epithelium could serve as cancer stem cells in tongue cancer.

    PubMed

    Tanaka, Toshihiro; Atsumi, Naho; Nakamura, Naohiro; Yanai, Hirotsugu; Komai, Yoshihiro; Omachi, Taichi; Tanaka, Kiyomichi; Ishigaki, Kazuhiko; Saiga, Kazuho; Ohsugi, Haruyuki; Tokuyama, Yoko; Imahashi, Yuki; Hisha, Hiroko; Yoshida, Naoko; Kumano, Keiki; Okazaki, Kazuichi; Ueno, Hiroo

    2016-12-22

    We recently reported that the polycomb complex protein Bmi1 is a marker for lingual epithelial stem cells (LESCs), which are involved in the long-term maintenance of lingual epithelial tissue in the physiological state. However, the precise role of LESCs in generating tongue tumors and Bmi1-positive cell lineage dynamics in tongue cancers are unclear. Here, using a mouse model of chemically (4-nitroquinoline-1-oxide: 4-NQO) induced tongue cancer and the multicolor lineage tracing method, we found that each unit of the tumor was generated by a single cell and that the assembly of such cells formed a polyclonal tumor. Although many Bmi1-positive cells within the tongue cancer specimens failed to proliferate, some proliferated continuously and supplied tumor cells to the surrounding area. This process eventually led to the formation of areas derived from single cells after 1-3 months, as determined using the multicolor lineage tracing method, indicating that such cells could serve as cancer stem cells. These results indicate that LESCs could serve as the origin for tongue cancer and that cancer stem cells are present in tongue tumors.

  15. Bmi1-positive cells in the lingual epithelium could serve as cancer stem cells in tongue cancer

    PubMed Central

    Tanaka, Toshihiro; Atsumi, Naho; Nakamura, Naohiro; Yanai, Hirotsugu; Komai, Yoshihiro; Omachi, Taichi; Tanaka, Kiyomichi; Ishigaki, Kazuhiko; Saiga, Kazuho; Ohsugi, Haruyuki; Tokuyama, Yoko; Imahashi, Yuki; Hisha, Hiroko; Yoshida, Naoko; Kumano, Keiki; Okazaki, Kazuichi; Ueno, Hiroo

    2016-01-01

    We recently reported that the polycomb complex protein Bmi1 is a marker for lingual epithelial stem cells (LESCs), which are involved in the long-term maintenance of lingual epithelial tissue in the physiological state. However, the precise role of LESCs in generating tongue tumors and Bmi1-positive cell lineage dynamics in tongue cancers are unclear. Here, using a mouse model of chemically (4-nitroquinoline-1-oxide: 4-NQO) induced tongue cancer and the multicolor lineage tracing method, we found that each unit of the tumor was generated by a single cell and that the assembly of such cells formed a polyclonal tumor. Although many Bmi1-positive cells within the tongue cancer specimens failed to proliferate, some proliferated continuously and supplied tumor cells to the surrounding area. This process eventually led to the formation of areas derived from single cells after 1–3 months, as determined using the multicolor lineage tracing method, indicating that such cells could serve as cancer stem cells. These results indicate that LESCs could serve as the origin for tongue cancer and that cancer stem cells are present in tongue tumors. PMID:28004815

  16. Twist1-positive epithelial cells retain adhesive and proliferative capacity throughout dissemination

    PubMed Central

    Shamir, Eliah R.; Coutinho, Kester; Georgess, Dan; Auer, Manfred

    2016-01-01

    ABSTRACT Dissemination is the process by which cells detach and migrate away from a multicellular tissue. The epithelial-to-mesenchymal transition (EMT) conceptualizes dissemination in a stepwise fashion, with downregulation of E-cadherin leading to loss of intercellular junctions, induction of motility, and then escape from the epithelium. This gain of migratory activity is proposed to be mutually exclusive with proliferation. We previously developed a dissemination assay based on inducible expression of the transcription factor Twist1 and here utilize it to characterize the timing and dynamics of intercellular adhesion, proliferation and migration during dissemination. Surprisingly, Twist1+ epithelium displayed extensive intercellular junctions, and Twist1– luminal epithelial cells could still adhere to disseminating Twist1+ cells. Although proteolysis and proliferation were both observed throughout dissemination, neither was absolutely required. Finally, Twist1+ cells exhibited a hybrid migration mode; their morphology and nuclear deformation were characteristic of amoeboid cells, whereas their dynamic protrusive activity, pericellular proteolysis and migration speeds were more typical of mesenchymal cells. Our data reveal that epithelial cells can disseminate while retaining competence to adhere and proliferate. PMID:27402962

  17. Analysis of Serial Isolates of mcr-1- positive Escherichia coli Reveals a Highly Active ISApl1 Transposon.

    PubMed

    Snesrud, Erik; Ong, Ana C; Corey, Brendan; Kwak, Yoon I; Clifford, Robert; Gleeson, Todd; Wood, Shannon; Whitman, Timothy J; Lesho, Emil P; Hinkle, Mary; Mc Gann, Patrick

    2017-02-21

    The emergence of the transferable colistin resistance gene mcr-1 is of global concern. The insertion sequence (IS) Apl1 is a key component in the mobilization of this gene, but its role remains poorly understood.Six Escherichia coli were cultured from the same patient over one month in Germany and the USA after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli were collected during follow-up surveillance nine months later. All isolates were analyzed with whole genome sequencing (WGS).WGS revealed that the six initial isolates were composed of two distinct strains; an initial ST-617 E.coli harboring mcr-1and a second, unrelated mcr-1-negative ST-32 E. coli that emerged 2 weeks after hospitalization. Follow-up swabs 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. Mcr-1 was associated with a single copy of ISApl1 located on a 64.5 Kb IncI2 plasmid that shared >95% homology to other mcr-1 IncI2 plasmids. ISApl1 copy number ranged from 2 in the first isolate to 6 in the final isolate, but movement was independent of mcr-1 Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress response, arginine catabolism and L-arabinose utilization.These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveals that under certain conditions ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.

  18. Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors.

    PubMed

    Awad, Mark M; Jones, Robert E; Liu, Hongye; Lizotte, Patrick H; Ivanova, Elena V; Kulkarni, Meghana; Herter-Sprie, Grit S; Liao, Xiaoyun; Santos, Abigail A; Bittinger, Mark A; Keogh, Lauren; Koyama, Shohei; Almonte, Christina; English, Jessie M; Barlow, Julianne; Richards, William G; Barbie, David A; Bass, Adam J; Rodig, Scott J; Hodi, F Stephen; Wucherpfennig, Kai W; Jänne, Pasi A; Sholl, Lynette M; Hammerman, Peter S; Wong, Kwok-Kin; Bueno, Raphael

    2016-12-01

    PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45(+) immune cells, a significantly higher proportion of infiltrating CD3(+) T cells, and a significantly higher percentage of CD3(+) cells displaying the activated HLA-DR(+)/CD38(+) phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8(+) T cells, a higher fraction of FOXP3(+)/CD4(+) Tregs, and increased expression of PD-1 and TIM-3 on CD4(+) and CD8(+) T cells. Double-positive PD-1(+)/TIM-3(+) CD8(+) T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3(+) T cells and PD-1(+)/TIM-3(+) CD8(+) T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR.

  19. Infiltration of PD-1-positive cells in combination with tumor site PD-L1 expression is a positive prognostic factor in cutaneous angiosarcoma.

    PubMed

    Honda, Yuki; Otsuka, Atsushi; Ono, Sachiko; Yamamoto, Yosuke; Seidel, Judith A; Morita, Satoshi; Hirata, Masahiro; Kataoka, Tatsuki R; Takenouchi, Tatsuya; Fujii, Kazuyasu; Kanekura, Takuro; Okubo, Yuko; Takahashi, Kenzo; Yanagi, Teruki; Hoshina, Daichi; Hata, Hiroo; Abe, Riichiro; Fujimura, Taku; Funakoshi, Takeru; Yoshino, Koji; Masuzawa, Mamiko; Amoh, Yasuyuki; Tanaka, Ryota; Fujisawa, Yasuhiro; Honda, Tetsuya; Kabashima, Kenji

    2017-01-01

    Cutaneous angiosarcoma (CAS) is a malignant sarcoma with poor prognosis. Programmed cell death-1 (PD-1)/programmed cell death-1 ligand-1 (PD-L1) expression reflects antitumor immunity, and is associated with patient prognosis in various cancers. The purpose of this study is to investigate the relationship between PD-1/PD-L1 expression and CAS prognosis. CAS cases (n = 106) were immunohistochemically studied for PD-L1 and PD-1 expression, and the correlation with patient prognosis was analyzed. PD-L1 expression was assessed by flow cytometry on three CAS cell lines with or without IFNγ stimulation. A total of 30.2% of patients' samples were positive for PD-L1, and 17.9% showed a high infiltration of PD-1-positive cells. Univariate analysis showed a significant relationship between a high infiltration of PD-1-positive cells with tumor site PD-L1 expression and favorable survival in stage 1 patients (p = 0.014, log-rank test). Multivariable Cox-proportional hazard regression analysis also showed that patients with a high infiltration of PD-1-positive cells with tumor site PD-L1 expression were more likely to have favorable survival, after adjustment with possible confounders (hazard ratio (HR) = 0.38, p = 0.021, 95% confidence interval (CI) 0.16-0.86). Immunofluorescence staining of CAS samples revealed that PD-L1-positive cells were adjacent to PD-1-positive cells and/or tumor stroma with high IFNγ expression. In vitro stimulation with IFNγ increased PD-L1 expression in two out of three established CAS cell lines. Our results suggest that PD-1/PD-L1 expression is related to CAS progression, and the treatment with anti-PD-1 antibodies could be a new therapeutic option for CAS.

  20. Decreased PD-1 positive blood follicular helper T cells in patients with psoriasis.

    PubMed

    Shin, Dongyun; Kim, Dae Suk; Kim, Sung Hee; Je, Jung Hwan; Kim, Hee Ju; Young Kim, Do; Kim, Soo Min; Lee, Min-Geol

    2016-10-01

    Follicular helper T (Tfh) cells are recently characterized subset of helper T cells, which are initially found in the germinal centers of B cell follicles. The major role of Tfh cells is helping B cell activation and antibody production during humoral immunity. Recently, blood Tfh cells were shown to be associated with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid and psoriasis. There is only one study which investigated Tfh cells in psoriasis patients. Therefore, in this study, we evaluated and analyzed blood Tfh cells in Korean patients with psoriasis. A total of 28 psoriasis patients and 16 healthy controls were enrolled. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells were decreased in patients with psoriasis compared to healthy controls. CD4(+)CXCR5(+) T cells and CXCR5(+)ICOS(+) Tfh cells did not show differences. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells in psoriasis patients negatively correlated with erythrocyte sedimentation rate and positively correlated with disease duration. The absolute number of CXCR5(+)ICOS(+) Tfh cells also showed positive correlation with disease duration. However, the subpopulations of Tfh cells did not correlate with Psoriasis Area and Severity Index. Serum interleukin-21 level was significantly increased in psoriasis patients compared to healthy controls, however, its level did not correlate with clinical and experimental parameters of psoriasis patients. These findings suggest the decreased function of Tfh cells in psoriasis, which could result in attenuated B cell immune responses in the pathogenesis of psoriasis. However, further investigations are necessary to confirm the function of Tfh cells in psoriasis vulgaris.

  1. 18O proteomics reveal increased Human Apolipoprotein CIII in Hispanic HIV-1 positive women with HAART that use cocaine

    PubMed Central

    Zenón, Frances; Jorge, Inmaculada; Cruz, Ailed; Suarez, Erick; Segarra, Annabell C.; Vázquez, Jesús; Meléndez, Loyda M.; Serrano, Horacio

    2016-01-01

    Purpose Drug abuse is a major risk factor in the development and progression of HIV-1. This study defines the alterations in the plasma proteome of HIV-1 infected women that use cocaine. Experimental Design Plasma samples from 12 HIV-seropositive Hispanic women under antiretroviral therapy were selected for this study. Six sample pairs were matched between non-drug users and cocaine users. After IgG and albumin depletion, SDS-PAGE, and in-gel digestion, peptides from non-drug users and cocaine users were labeled with 16O and 18O respectively and subjected to LC-MS/MS and quantitation using Proteome Discover and QuiXoT softwares and validated by ELISA. Results A total of 1,015 proteins were identified at 1% FDR. Statistical analyses revealed 13 proteins with significant changes between the two groups, cocaine and non-cocaine users (p<0.05). The great majority pertained to protection defense function and the rest pertained to transport, homeostatic, regulation, and binding of ligands. Apolipoprotein CIII was increased in plasma of HIV+ Hispanic women positive for cocaine compared to HIV+ non-drug users (p<0.05). Conclusions and clinical relevance Increased human Apolipoprotein CIII warrants that these patients be carefully monitored to avoid the increased risk of cardiovascular events associated with HIV, HAART and cocaine use. PMID:26255783

  2. Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina.

    PubMed

    Dvoriantchikova, Galina; Perea-Martinez, Isabel; Pappas, Steve; Barry, Ariel Faye; Danek, Dagmara; Dvoriantchikova, Xenia; Pelaez, Daniel; Ivanov, Dmitry

    2015-01-01

    The oscillatory expression of Notch signaling in neural progenitors suggests that both repressors and activators of neural fate specification are expressed in the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification, we hypothesized that genes encoding photoreceptor and ganglion cell fate activators would be highly expressed in Notch1 receptor-bearing (Notch1+) progenitors, directing these cells to differentiate into photoreceptors or into ganglion cells when Notch1 activity is diminished. To identify these genes, we used microarray analysis to study expression profiles of whole retinas and isolated from them Notch1+ cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1+ cells, we utilized immunomagnetic cell separation. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of Notch3 signaling in ganglion cell differentiation. Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1+ cells grouped near the same developmental stage retina cluster. At E14, we found higher expression of repressors (Notch1, Hes5) and activators (Dll3, Atoh7, Otx2) of neuronal differentiation in Notch1+ cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1+ cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1+ cells at P0. We also observed that retinas of wild type animals had only 14% (P < 0.05) more ganglion cells compared to Notch3KO mice. Since this number is relatively small and Notch1 has been shown to contribute to ganglion cell fate specification, we suggested that Notch1 signaling may play a more significant role in RGC development than the Notch3 signaling cascade. Finally, our findings suggest that Notch1+ progenitors--since they heavily express both pro-ganglion cell (Atoh7) and pro

  3. Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues.

    PubMed

    Di Rocco, Giuliana; Tritarelli, Alessandra; Toietta, Gabriele; Gatto, Ilaria; Iachininoto, Maria Grazia; Pagani, Francesca; Mangoni, Antonella; Straino, Stefania; Capogrossi, Maurizio C

    2008-02-01

    At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

  4. A cyclin D1-positive diffuse large B-cell lymphoma of germinal center B-cell-like subtype in the right tonsil

    PubMed Central

    Jiang, Changrui; Shi, Xiuying; Fan, Chuifeng

    2017-01-01

    Abstract Introduction: Cyclin D1-positive tumor cells are commonly found in mantle cell lymphoma but they are very rare in diffuse large B-cell lymphoma. Clinical findings/Patient concerns: Here we present a rare case of cyclin D1-positive diffuse large B-cell lymphoma in the right tonsil of a 50-year-old man. Computed tomographic imaging detected a mass, about 2.5 cm × 1.8 cm in size, in the left side of the oropharynx. Diagnoses: Microscopically, the tumor cells were located under the pharyngeal mucosa and diffusely arranged. The tumor cells were large, with marked nuclear atypia. On performing immunohistochemistry, the tumor cells showed diffuse positive staining for CD10, CD20, cyclin D1, and Pax-5, and negative staining for CD3, CD15, CD30, CD56, and CK. Bcl-6 and Mum-1 expression were observed in 60% and 80% of tumor cells, respectively. The tumor Ki67 index was about 60%. Based on these findings, The tumor was diagnosed as a rare cyclin D1-positive diffuse large B-cell lymphoma rather than a mantle cell lymphoma. Conclusion: Cyclin D1-positive large B-cell lymphoma is rare, but as large B-cell lymphoma is a common type of lymphoma, cyclin D1-positive large B-cell lymphoma should be considered a major possibility during differential diagnosis, including in the tonsils. PMID:28296741

  5. Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

    PubMed

    Li, Xiaobing; Saitoh, Shin-Ichiroh; Shibata, Takuma; Tanimura, Natsuko; Fukui, Ryutaro; Miyake, Kensuke

    2015-02-01

    Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs.

  6. DOXIL when combined with Withaferin A (WFA) targets ALDH1 positive cancer stem cells in ovarian cancer.

    PubMed

    Kakar, Sham S; Worth, Christopher A; Wang, Zhenglong; Carter, Kelsey; Ratajczak, Mariusz; Gunjal, Pranesh

    Ovarian cancer is a highly aggressive and deadly disease. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately, after few months of initial treatment, tumor relapse occurs due to platinum-resistance. DOXIL (liposomal preparation of doxorubicin) is a choice of drug for recurrent ovarian cancer. However, its response rate is very low and is accompanied by myocardial toxicity. Resistance to chemotherapy and recurrence of cancer is primarily attributed to the presence of cancer stem cells (CSCs), a small population of cells present in cancer. Effect of DOXIL and withaferin A (WFA), both alone and in combination, was investigated on cell proliferation of ovarian cancer cell line A2780 and tumor growth in SCID mice bearing i.p. ovarian tumors. ALDH1 cells were isolated from A2780 using cell sorter, and effect of DOXIL and WFA both alone and in combination on tumorigenic function of ALDH1 was studied using spheroids formation assays in vitro. Western blots were performed to examine the expression of ALDH1 and Notch 1 genes. In our studies, we showed, for the first time, that DOXIL when combined with withaferin A (WFA) elicits synergistic effect on inhibition of cell proliferation of ovarian cancer cells and inhibits the expression of ALDH1 protein, a marker for ALDH1 positive cancer stem cells (CSCs), and Notch1, a signaling pathway gene required for self-renewal of CSCs. Inhibition of expression of both ALDH1 and Notch1 genes by WFA was found to be dose dependent, whereas DOXIL (200 nM) was found to be ineffective. SCID mice, bearing i.p. ovarian tumors, were treated with a small dose of DOXIL (2 mg/kg) in combination with a sub-optimal dose of WFA (2 mg/kg) which resulted in a highly significant (60% to 70%) reduction in tumor growth, and complete inhibition of metastasis

  7. Ultrastructural identification of Ricinus communis agglutinin-1 positive cells in primary dissociated cell cultures of human embryonic brain.

    PubMed

    Bobryshev, Y; Ashwell, K

    1994-12-01

    While Ricinus communis agglutinin 1 (RCA-1) can be used as a specific marker to study the development and differentiation of microglial cells in human embryogenesis, little is known about the structural heterogeneity and nature of RCA-1+ cells. To analyse the structural peculiarities of RCA-1+ cells, we have used primary dissociated cultures of human embryonic brain. These have been used as models for investigating many of the aspects of central nervous system (CNS) HIV infection. We have shown that primary dissociated cultures from human embryos as young as 10 weeks gestation contain RCA-1+ cells. The RCA-1+ cells exist in two forms, those without (type I) and those with (type II) processes. The former have a poorly developed ultrastructure, while the latter have well developed ultrastructural features, such as rough endoplasmic reticulum with short cisternae, abundant ribosomes, mitochondria, lysosomes and vacuoles. Furthermore, some of these cells with processes have well developed cytoskeletal features. In this paper, the classification of RCA-1+ cells of embryonic human brain is considered and their morphology compared to microglia identified in rodent CNS.

  8. Selective Toxicity of NSC 73306 in MDR1-positive cells as a New Strategy to Circumvent Multidrug Resistance in Cancer

    PubMed Central

    Ludwig, Joseph A.; Martin, Scott E.; Chu, Benjamin F.; Cardarelli, Carol; Sauna, Zuben E.; Caplen, Natasha J.; Fales, Henry M.; Ambudkar, Suresh V.; Weinstein, John N.

    2006-01-01

    ATP-binding cassette (ABC) proteins include the best known mediators of resistance to anticancer drugs. In particular, ABCB1 (MDR1/P-gp) extrudes many types of drugs from cancer cells, thereby conferring resistance to those agents. Attempts to overcome P-gp-mediated drug resistance using specific inhibitors of P-gp has had limited success, and has faced many therapeutic challenges. As an alternative approach to using P-gp inhibitors, we characterize a thiosemicarbazone derivative (NSC73306) identified in a generic screen as a compound that exploits, rather than suppresses, P-gp function to induce cytotoxicity. Cytotoxic activity of NSC73306 was evaluated in vitro using human epidermoid, ovarian, and colon cancer cell lines expressing various levels of P-gp. Our findings suggest that cells become hypersensitive to NSC73306 in proportion to the increased P-gp function and multidrug resistance (MDR). Abrogation of both sensitivity to NSC73306 and resistance to P-gp substrate anticancer agents occurred with specific inhibition of P-gp function using either a P-gp inhibitor (PSC833, XR9576) or RNA interference (RNAi), suggesting that cytotoxicity was linked to MDR1 function, not to other, nonspecific factors arising during the generation of resistant or transfected cells. Molecular characterization of cells selected for resistance to NSC73306 revealed loss of P-gp expression and consequent loss of the MDR phenotype. Although hypersensitivity to NSC73306 required functional expression of P-gp, biochemical assays revealed no direct interaction between NSC73306 and P-gp. This work demonstrates that NSC73306 kills cells with intrinsic or acquired P-gp-induced MDR and indirectly acts to eliminate resistance to MDR1 substrates. PMID:16651436

  9. Monoclonal antibody conjugated magnetic nanoparticles could target MUC-1-positive cells in vitro but not in vivo.

    PubMed

    Shanehsazzadeh, Saeed; Gruettner, Cordula; Lahooti, Afsaneh; Mahmoudi, Morteza; Allen, Barry J; Ghavami, Mahdi; Daha, Fariba Johari; Oghabian, Mohammad Ali

    2015-01-01

    MUC1 antigen is recognized as a high-molecular-weight glycoprotein that is unexpectedly over-expressed in human breast and other carcinomas. In contrast, C595 a monoclonal antibody (mAb) against the protein core of the human urinary epithelial machine, is commonly expressed in breast carcinomas. The aim of this study was to conjugate ultra-small super paramagnetic iron oxide nanoparticles (USPIO) with C595 mAb, in order to detect in vivo MUC1 expression. A dual contrast agent (the C595 antibody-conjugated USPIO labeled with 99mTc) was prepared for targeted imaging and therapy of anti-MUC1-expressing cancers. The C595 antibody-conjugated USPIO had good stability and reactivity in the presence of blood plasma at 37 °C. No significant differences were observed in immunoreactivity results between conjugated and nonconjugated nanoparticles. The T1 and T2 measurements show >79 and 29% increments (for 0.02 mg/ml iron concentrations) in T1 and T2 values for USPIO-C595 in comparison with USPIO, respectively. The nanoprobes showed the interesting targeting capability of finding the MUC1-positive cell line in vitro. However, we found disappointing in vivo results (i.e. very low accumulation of nanoprobes in the targeted site while >80% of the injected dose per gram was taken up by the liver and spleen), not only due to the coverage of targeting site by protein corona but also because of absorption of opsonin-based proteins at the surface of nanoprobes.

  10. ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program.

    PubMed

    Charafe-Jauffret, Emmanuelle; Ginestier, Christophe; Bertucci, François; Cabaud, Olivier; Wicinski, Julien; Finetti, Pascal; Josselin, Emmanuelle; Adelaide, José; Nguyen, Tien-Tuan; Monville, Florence; Jacquemier, Jocelyne; Thomassin-Piana, Jeanne; Pinna, Guillaume; Jalaguier, Aurélie; Lambaudie, Eric; Houvenaeghel, Gilles; Xerri, Luc; Harel-Bellan, Annick; Chaffanet, Max; Viens, Patrice; Birnbaum, Daniel

    2013-12-15

    Cancer stem-like cells (CSC) have been widely studied, but their clinical relevance has yet to be established in breast cancer. Here, we report the establishment of primary breast tumor-derived xenografts (PDX) that encompass the main diversity of human breast cancer and retain the major clinicopathologic features of primary tumors. Successful engraftment was correlated with the presence of ALDH1-positive CSCs, which predicted prognosis in patients. The xenografts we developed showed a hierarchical cell organization of breast cancer with the ALDH1-positive CSCs constituting the tumorigenic cell population. Analysis of gene expression from functionally validated CSCs yielded a breast CSC signature and identified a core transcriptional program of 19 genes shared with murine embryonic, hematopoietic, and neural stem cells. This generalized stem cell program allowed the identification of potential CSC regulators, which were related mainly to metabolic processes. Using an siRNA genetic screen designed to target the 19 genes, we validated the functional role of this stem cell program in the regulation of breast CSC biology. Our work offers a proof of the functional importance of CSCs in breast cancer, and it establishes the reliability of PDXs for use in developing personalized CSC therapies for patients with breast cancer.

  11. EVI1-mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells.

    PubMed

    De Weer, An; Van der Meulen, Joni; Rondou, Pieter; Taghon, Tom; Konrad, Torsten A; De Preter, Katleen; Mestdagh, Pieter; Van Maerken, Tom; Van Roy, Nadine; Jeison, Marta; Yaniv, Isaac; Cauwelier, Barbara; Noens, Lucien; Poirel, Hélène-Antoine; Vandenberghe, Peter; Lambert, Frédéric; De Paepe, Anne; Sánchez, Maria García; Odero, Maria; Verhasselt, Bruno; Philippé, Jan; Vandesompele, Joke; Wieser, Rotraud; Dastugue, Nicole; Van Vlierberghe, Pieter; Poppe, Bruce; Speleman, Frank

    2011-08-01

    Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup.

  12. Accumulation of ALDH1-positive cells after neoadjuvant chemotherapy predicts treatment resistance and prognosticates poor outcome in ovarian cancer.

    PubMed

    Ayub, Tiyasha H; Keyver-Paik, Mignon-Denise; Debald, Manuel; Rostamzadeh, Babak; Thiesler, Thore; Schröder, Lars; Barchet, Winfried; Abramian, Alina; Kaiser, Christina; Kristiansen, Glen; Kuhn, Walther; Kübler, Kirsten

    2015-06-30

    Although ovarian cancer is a highly chemosensitive disease, it is only infrequently cured. One of the major reasons lies in the presence of drug-resistant cancer stem-like cells, sufficient to fuel recurrence. We phenotyped cancer stem-like cells by flow cytometry and immunohistochemistry in 55 matched samples before and after taxane/platinum-based neoadjuvant chemotherapy. All used markers of stemness (ALDH1, CD24, CD117, CD133) isolated low frequencies of malignant cells. ALDH1 was the most valuable marker for tracking stemness in vivo. The enrichment of ALDH1 expression after treatment was associated with a poor response to chemotherapy, with platinum resistance and independently prognosticated unfavorable outcome. Our results suggest that increased ALDH1 expression after treatment identifies patients with aggressive tumor phenotypes.

  13. Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

    PubMed Central

    Wu, Yuntao

    2010-01-01

    Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells. PMID:20972394

  14. Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

    PubMed

    Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

    2013-04-18

    We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

  15. Sphingosylphosphorylcholine promotes the differentiation of resident Sca-1 positive cardiac stem cells to cardiomyocytes through lipid raft/JNK/STAT3 and β-catenin signaling pathways.

    PubMed

    Li, Wenjing; Liu, Honghong; Liu, Pingping; Yin, Deling; Zhang, Shangli; Zhao, Jing

    2016-07-01

    Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake.

  16. MAPK/ERK and Wnt/{beta}-Catenin pathways are synergistically involved in proliferation of Sca-1 positive hepatic progenitor cells

    SciTech Connect

    Jin, Caixia; Samuelson, Lisa; Cui, Cai-Bin; Sun, Yangzhong; Gerber, David A.

    2011-06-17

    Highlights: {yields} Activation of MAPK/ERK pathway with epidermal growth factor (EGF) significantly increased Sca-1{sup +} HPC proliferation and colony formation. {yields} Activation of either IL-6/STAT3 or Wnt/{beta}-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. {yields} Wnt/{beta}-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation and maintain long-term HPCs in vitro. -- Abstract: Hepatic progenitor cells (HPCs) persist in adulthood and have the potential to play a major role in regenerating diseased liver. However, the signaling pathways that both directly and indirectly regulate HPCs' self-renewal and differentiation remain elusive. Previously, we identified a bipotent, stem cell antigen-1 (Sca-1) positive HPC population from naive adult liver tissue. In the present study, we aimed to investigate the involvement of various signaling pathways in Sca-1{sup +} HPC proliferation. Epidermal growth factor (EGF) supplementation shows a significant increase in Sca-1{sup +} HPC proliferation and colony formation while stimulating phosphorylation of ERK1/2 and activating the induction of Cyclin D1. There were no demonstrable effects of EGF on Akt. The MEK inhibitor, PD0325901, inhibits proliferation and ERK1/2 phosphorylation while also suppressing the expression of Cyclin D1. In addition, activation of either IL-6/STAT3 or Wnt/{beta}-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. The Wnt/{beta}-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation ratio and maintain long-term HPC in vitro. The data indicates that the MAPK/ERK pathway is both essential and critical for HPC proliferation, and the Wnt signaling pathway is not sufficient, while it works synergistically with the MAPK/ERK signaling pathway to promote HPC proliferation.

  17. Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors.

    PubMed

    McCormack, Emmet; Adams, Katherine J; Hassan, Namir J; Kotian, Akhil; Lissin, Nikolai M; Sami, Malkit; Mujić, Maja; Osdal, Tereza; Gjertsen, Bjørn Tore; Baker, Deborah; Powlesland, Alex S; Aleksic, Milos; Vuidepot, Annelise; Morteau, Olivier; Sutton, Deborah H; June, Carl H; Kalos, Michael; Ashfield, Rebecca; Jakobsen, Bent K

    2013-04-01

    NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.

  18. Epigallocatechin-3-gallate inhibits tax-dependent activation of nuclear factor kappa B and of matrix metalloproteinase 9 in human T-cell lymphotropic virus-1 positive leukemia cells.

    PubMed

    Harakeh, Steve; Diab-Assaf, Mona; Azar, Rania; Hassan, Hani Mutlak Abdulla; Tayeb, Safwan; Abou-El-Ardat, Khalil; Damanhouri, Ghazi Abdullah; Qadri, Ishtiaq; Abuzenadah, Adel; Chaudhary, Adeel; Kumosani, Taha; Niedzwiecki, Aleksandra; Rath, Mathias; Yacoub, Haitham; Azhar, Esam; Barbour, Elie

    2014-01-01

    Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol molecule from green tea and is known to exhibit antioxidative as well as tumor suppressing activity. In order to examine EGCG tumor invasion and suppressing activity against adult T-cell leukemia (ATL), two HTLV-1 positive leukemia cells (HuT-102 and C91- PL) were treated with non-cytotoxic concentrations of EGCG for 2 and 4 days. Proliferation was significantly inhibited by 100 μM at 4 days, with low cell lysis or cytotoxicity. HTLV-1 oncoprotein (Tax) expression in HuT- 102 and C91-PL cells was inhibited by 25 μM and 125 μM respectively. The same concentrations of EGCG inhibited NF-kB nuclearization and stimulation of matrix metalloproteinase-9 (MMP-9) expression in both cell lines. These results indicate that EGCG can inhibit proliferation and reduce the invasive potential of HTLV-1- positive leukemia cells. It apparently exerted its effects by suppressing Tax expression, manifested by inhibiting the activation of NF-kB pathway and induction of MMP-9 transcription in HTLV-1 positive cells.

  19. Intravenous renal cell transplantation with SAA1-positive cells prevents the progression of chronic renal failure in rats with ischemic-diabetic nephropathy.

    PubMed

    Kelly, Katherine J; Zhang, Jizhong; Han, Ling; Wang, Mingsheng; Zhang, Shaobo; Dominguez, Jesus H

    2013-12-15

    Diabetic nephropathy, the most common cause of progressive chronic renal failure and end-stage renal disease, has now reached global proportions. The only means to rescue diabetic patients on dialysis is renal transplantation, a very effective therapy but severely limited by the availability of donor kidneys. Hence, we tested the role of intravenous renal cell transplantation (IRCT) on obese/diabetic Zucker/SHHF F1 hybrid (ZS) female rats with severe ischemic and diabetic nephropathy. Renal ischemia was produced by bilateral renal clamping of the renal arteries at 10 wk of age, and IRCT with genetically modified normal ZS male tubular cells was given intravenously at 15 and 20 wk of age. Rats were euthanized at 34 wk of age. IRCT with cells expressing serum amyloid A had strong and long-lasting beneficial effects on renal function and structure, including tubules and glomeruli. However, donor cells were found engrafted only in renal tubules 14 wk after the second infusion. The results indicate that IRCT with serum amyloid A-positive cells is effective in preventing the progression of chronic kidney disease in rats with diabetic and ischemic nephropathy.

  20. Monitoring PD-L1 positive circulating tumor cells in non-small cell lung cancer patients treated with the PD-1 inhibitor Nivolumab

    PubMed Central

    Nicolazzo, Chiara; Raimondi, Cristina; Mancini, MariaLaura; Caponnetto, Salvatore; Gradilone, Angela; Gandini, Orietta; Mastromartino, Maria; del Bene, Gabriella; Prete, Alessandra; Longo, Flavia; Cortesi, Enrico; Gazzaniga, Paola

    2016-01-01

    Controversial results on the predictive value of programmed death ligand 1 (PD-L1) status in lung tumor tissue for response to immune checkpoint inhibitors do not allow for any conclusive consideration. Liquid biopsy might allow real-time sampling of patients for PD-L1 through the course of the disease. Twenty-four stage IV NSCLC patients included in the Expanded Access Program with Nivolumab were enrolled. Circulating tumor cells (CTCs) were analyzed by CellSearch with anti-human B7-H1/PD-L1 PE-conjugated antibody. PD-L1 expressing CTCs were assessed at baseline, at 3 and 6 months after starting therapy, and correlated with outcome. At baseline and at 3 months of treatment, the presence of CTCs and the expression of PD-L1 on their surface were found associated to poor patients outcome. Nevertheless, the high frequency of PD-L1 expressing CTCs hampered to discriminate the role of PD-L1 in defining prognosis. Conversely although CTCs were found in all patients 6 months after treatment, at this time patients could be dichotomized into two groups based PD-L1 expression on CTCs. Patients with PD-L1 negative CTCs all obtained a clinical benefit, while patients with PD-L1 (+) CTCs all experienced progressive disease. This suggests that the persistence of PD-L1(+) CTCs might mirror a mechanism of therapy escape. PMID:27553175

  1. Influence of Differently Licensed KIR2DL1-Positive Natural Killer Cells in Transplant Recipients with Acute Leukemia: A Japanese National Registry Study.

    PubMed

    Arima, Nobuyoshi; Nakamura, Fumiaki; Yabe, Toshio; Tanaka, Junji; Fuji, Shigeo; Ohashi, Kazuteru; Fukuda, Takahiro; Miyamura, Koichi; Iwato, Koji; Eto, Tetsuya; Mori, Takehiko; Kobayashi, Naoki; Hoshino, Takumi; Kato, Chiaki; Kanamori, Heiwa; Nakamae, Hirohisa; Atsuta, Yoshiko; Morishima, Yasuo; Kanda, Yoshinobu

    2016-03-01

    Licensing by self MHC class I ligands is required for proper natural killer (NK) cell response. NK cells with inhibitory killer cell immunoglobulin-like receptors for nonself MHC exhibit transient alloreactivity after hematopoietic stem cell transplantation (HSCT). We analyzed 3866 recipients in the Japan national registry who underwent their first allogeneic HSCT for acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) from HLA-A, -B, and -DRB1 allele-genomatched unrelated donors. By classifying them into 5 independent groups based on HLA-C group matching and assumed donor NK cell status, we found that for HLA-C-matched HSCT for AML in HLA-C1/C1 recipients, in whom transient alloreactivity against HLA-C2-negative leukemic cells was expected, the relapse rate was significantly lower than it was in HLA-C-matched HSCT for AML in HLA-C1/C2 recipients (hazard ratio [HR], .72; P = .011). This difference was not observed in HLA-C-matched HSCT for ALL. Compared with HLA-C-matched HSCT, significantly higher mortality was observed in HLA-C1/C1 AML patients who received transplants from HLA-C-mismatched HLA-C1/C1 donors (HR, 1.37; P = .001) and in HLA-C1/C1 ALL patients who received transplants from HLA-C2-positive donors (HR, 2.13; P = .005). In conclusion, donor selection based on leukemic subtype and donor HLA-C group matching improves transplantation outcome after HLA-C-mismatched HSCT.

  2. Effects of BMAL1-SIRT1-positive cycle on estrogen synthesis in human ovarian granulosa cells: an implicative role of BMAL1 in PCOS.

    PubMed

    Zhang, Jiaou; Liu, Jiansheng; Zhu, Kai; Hong, Yan; Sun, Yun; Zhao, Xiaoming; Du, Yanzhi; Chen, Zi-Jiang

    2016-08-01

    Brain and muscle ARNT-like protein 1 (BMAL1) is necessary for fertility and has been found to be essential to follicle growth and steroidogenesis. Sirtuin1 (SIRT1) has been reported to interact with BMAL1 and function in a circadian manner. Evidence has shown that SIRT1 regulates aromatase expression in estrogen-producing cells. We aimed to ascertain if there is a relationship between polycystic ovary syndrome (PCOS) and BMAL1, and whether and how BMAL1 takes part in estrogen synthesis in human granulosa cells (hGCs). Twenty-four women diagnosed with PCOS and 24 healthy individuals undergoing assisted reproduction were studied. BMAL1 expression in their granulosa cells (GCs) was observed by quantitative real-time polymerase chain reaction (qRT-PCR). The level of expression in the PCOS group was lower than that of the group without PCOS (p < 0.05). We also analyzed estrogen synthesis and aromatase expression in KGN cell lines. Both were downregulated after BMAL1 and SIRT1 knock-down and, conversely, upregulated after overexpression treatments of these two genes in KGN cells. Both BMAL1 and SIRT1 had a mutually positive regulation, as did the phosphorylation of JNK. Furthermore, JNK overexpression increased estrogen synthesis activity and the expression levels of aromatase, BMAL1, and SIRT1. In KGN and hGCs, estrogen synthesis and aromatase expression were downregulated after treatment with JNK and SIRT1 inhibitors. In addition, BMAL1, SIRT1, and JNK expression levels were all downregulated. Our results demonstrate the effects of BMAL1 on estrogen synthesis in hGCs and suggest a BMAL1-SIRT1-JNK positive feedback cycle in this process, which points out an important role of BMAL1 in the development of PCOS.

  3. Peripheral myeloid-derived suppressor and T regulatory PD-1 positive cells predict response to neoadjuvant short-course radiotherapy in rectal cancer patients.

    PubMed

    Napolitano, Maria; D'Alterio, Crescenzo; Cardone, Eleonora; Trotta, Anna Maria; Pecori, Biagio; Rega, Daniela; Pace, Ugo; Scala, Dario; Scognamiglio, Giosuè; Tatangelo, Fabiana; Cacciapuoti, Carmela; Pacelli, Roberto; Delrio, Paolo; Scala, Stefania

    2015-04-10

    Short-course preoperative radiotherapy (SC-RT) followed by total mesorectal excision (TME) is one therapeutic option for locally advanced rectal cancer (LARC) patients. Since radio-induced DNA damage may affect tumor immunogenicity, Myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) were evaluated in 13 patients undergoing SC-RT and TME for LARC. Peripheral Granulocytic-MDSCs (G-MDSC) [LIN-/HLA-DR-/CD11b+/CD14-/CD15+/CD33+], Monocytic (M-MDSC) [CD14+/HLA-DR-/lowCD11b+/CD33+] and Tregs [CD4+/CD25hi+/FOXP3+- CTLA-4/PD1] basal value was significantly higher in LARC patients compared to healthy donors (HD). Peripheral MDSC and Tregs were evaluated at time 0 (T0), after 2 and 5 weeks (T2-T5) from radiotherapy; before surgery (T8) and 6-12 months after surgery (T9, T10). G-MDSC decreased at T5 and further at T8 while M-MDSC cells decreased at T5; Tregs reached the lowest value at T5. LARC poor responder patients displayed a major decrease in M-MDSC after SC-RT and an increase of Treg-PD-1. In this pilot study MDSCs and Tregs decrease during the SC-RT treatment could represent a biomarker of response in LARC patients. Further studies are needed to confirm that the deepest M-MDSC reduction and increase in Treg-PD1 cells within 5-8 weeks from the beginning of treatment could discriminate LARC patients poor responding to SC-RT.

  4. Effects of Intermittent Administration of Parathyroid Hormone (1-34) on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

    PubMed Central

    Wang, Xiaoxiao; Wang, Yanlan; Dai, Xubin; Chen, Tianyu; Yang, Fanqiao; Dai, Shuangye; Ou, Qianmin; Wang, Yan; Lin, Xuefeng

    2016-01-01

    Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs) may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1) has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH) has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+) and STRO-1(−) hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+) hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+) hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R) than STRO-1(−) hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+) hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+) hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+) hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis. PMID:27069479

  5. BDCA1-Positive Dendritic Cells (DCs) Represent a Unique Human Myeloid DC Subset That Induces Innate and Adaptive Immune Responses to Staphylococcus aureus Infection

    PubMed Central

    Zhang, Wei; Du, Jiang-yuan; Yu, Qing

    2014-01-01

    Staphylococcus aureus bloodstream infection (bacteremia) is a major cause of morbidity and mortality and places substantial cost burdens on health care systems. The role of peripheral blood dendritic cells (PBDCs) in the immune responses against S. aureus infection has not been well characterized. In this study, we demonstrated that BDCA1+ myeloid DCs (mDCs) represent a unique PBDC subset that can induce immune responses against S. aureus infection. BDCA1+ mDCs could engulf S. aureus and strongly upregulated the expression of costimulatory molecules and production of proinflammatory cytokines. Furthermore, BDCA1+ mDCs expressed high levels of major histocompatibility complex (MHC) class I and II molecules in response to S. aureus and greatly promoted proliferation and gamma interferon (IFN-γ) production in CD4 and CD8 T cells. Moreover, BDCA1+ mDCs expressed higher levels of Toll-like receptor 2 (TLR-2) and scavenger receptor A (SR-A) than those on CD16+ and BDCA3+ mDCs, and these two receptors were both required for the recognition of S. aureus and the subsequent activation of BDCA1+ mDCs. Finally, BDCA1+ mDC-mediated immune responses against S. aureus were dependent on MyD88 signaling pathways. These results demonstrate that human BDCA1+ mDCs represent a unique subset of mDCs that can respond to S. aureus to undergo maturation and activation and to induce Th1 and Tc1 immune responses. PMID:25114114

  6. ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.

    PubMed

    Flores-Rodriguez, Neftali; Kenwright, David A; Chung, Pei-Hua; Harrison, Andrew W; Stefani, Flavia; Waigh, Thomas A; Allan, Victoria J; Woodman, Philip G

    2015-02-15

    Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.

  7. Glial cell line-derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse.

    PubMed

    Zwick, Melissa; Davis, Brian M; Woodbury, C Jeffrey; Burkett, John N; Koerber, H Richard; Simpson, James F; Albers, Kathryn M

    2002-05-15

    Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.

  8. Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity.

    PubMed

    Chen, Renchao; Wu, Xiaoji; Jiang, Lan; Zhang, Yi

    2017-03-28

    The hypothalamus is one of the most complex brain structures involved in homeostatic regulation. Defining cell composition and identifying cell-type-specific transcriptional features of the hypothalamus is essential for understanding its functions and related disorders. Here, we report single-cell RNA sequencing results of adult mouse hypothalamus, which defines 11 non-neuronal and 34 neuronal cell clusters with distinct transcriptional signatures. Analyses of cell-type-specific transcriptomes reveal gene expression dynamics underlying oligodendrocyte differentiation and tanycyte subtypes. Additionally, data analysis provides a comprehensive view of neuropeptide expression across hypothalamic neuronal subtypes and uncover Crabp1(+) and Pax6(+) neuronal populations in specific hypothalamic sub-regions. Furthermore, we found food deprivation exhibited differential transcriptional effects among the different neuronal subtypes, suggesting functional specification of various neuronal subtypes. Thus, the work provides a comprehensive transcriptional perspective of adult hypothalamus, which serves as a valuable resource for dissecting cell-type-specific functions of this complex brain region.

  9. Leydig Cell Hyperplasia Revealed by Gynecomastia

    PubMed Central

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably. PMID:18660859

  10. Leydig cell hyperplasia revealed by gynecomastia.

    PubMed

    Tazi, Mohamed Fadl; Mellas, Soufiane; El Fassi, Mohamed Jamal; Farih, Moulay Hassan

    2008-01-01

    Leydig cell tumors are rare and represent 1% to 3% of all tumors of the testis. Leydig cell tumors affect males at any age, but there are 2 peak periods of incidence: between 5 and 10 years and between 25 and 35 years. Their main clinical presentation is a testicular mass associated with endocrinal manifestations that are variable according to age and appearance of the tumor. Our patient, a 17-year-old adolescent, presented with an isolated and painless hypertrophy of the right mammary gland. Clinical examination found gynecomastia and no testicular mass. Hormonal levels and tumor markers were normal. Testicular sonography showed an ovular and homogeneous right intratesticular mass 6 mm in diameter. We treated the patient with an inguinal right orchidectomy. The anatomopathological study found a nodule of Leydig cell hyperplasia. The patient recovered without recurrence at 8-month follow-up. The patient opted for mammoplasty 2 months after his orchidectomy rather than wait for the spontaneous gradual regression of his gynecomastia, which requires at least 1 year. Leydig cell hyperplasia manifests in the adult by signs of hypogonadism, most frequently gynecomastia. Although many teams prefer total orchidectomy because of the diagnostic difficulty associated with malignant forms, simple subcapsular orchidectomy should become the first-line treatment, provided it be subsequently followed by close surveillance, as it preserves maximum fertility, and these tumors usually resolve favorably.

  11. Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.

    PubMed

    Brusnahan, S K; McGuire, T R; Jackson, J D; Lane, J T; Garvin, K L; O'Kane, B J; Berger, A M; Tuljapurkar, S R; Kessinger, M A; Sharp, J G

    2010-01-01

    Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive.

  12. Single-cell chromatin accessibility reveals principles of regulatory variation.

    PubMed

    Buenrostro, Jason D; Wu, Beijing; Litzenburger, Ulrike M; Ruff, Dave; Gonzales, Michael L; Snyder, Michael P; Chang, Howard Y; Greenleaf, William J

    2015-07-23

    Cell-to-cell variation is a universal feature of life that affects a wide range of biological phenomena, from developmental plasticity to tumour heterogeneity. Although recent advances have improved our ability to document cellular phenotypic variation, the fundamental mechanisms that generate variability from identical DNA sequences remain elusive. Here we reveal the landscape and principles of mammalian DNA regulatory variation by developing a robust method for mapping the accessible genome of individual cells by assay for transposase-accessible chromatin using sequencing (ATAC-seq) integrated into a programmable microfluidics platform. Single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells in aggregate closely resemble accessibility profiles from tens of millions of cells and provide insights into cell-to-cell variation. Accessibility variance is systematically associated with specific trans-factors and cis-elements, and we discover combinations of trans-factors associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of cell cycle or transcription factor signalling evoke stimulus-specific changes in this observed variability. The pattern of accessibility variation in cis across the genome recapitulates chromosome compartments de novo, linking single-cell accessibility variation to three-dimensional genome organization. Single-cell analysis of DNA accessibility provides new insight into cellular variation of the 'regulome'.

  13. Macrophage characteristics of stem cells revealed by transcriptome profiling

    SciTech Connect

    Charriere, Guillaume M.; Cousin, Beatrice; Arnaud, Emmanuelle; Saillan-Barreau, Corinne; Andre, Mireille; Massoudi, Ali; Dani, Christian; Penicaud, Luc; Casteilla, Louis . E-mail: casteil@toulouse.inserm.fr

    2006-10-15

    We previously showed that the phenotypes of adipocyte progenitors and macrophages were close. Using functional analyses and microarray technology, we first tested whether this intriguing relationship was specific to adipocyte progenitors or could be shared with other progenitors. Measurements of phagocytic activity and gene profiling analysis of different progenitor cells revealed that the latter hypothesis should be retained. These results encouraged us to pursue and to confirm our analysis with a gold-standard stem cell population, embryonic stem cells or ESC. The transcriptomic profiles of ESC and macrophages were clustered together, unlike differentiated ESC. In addition, undifferentiated ESC displayed higher phagocytic activity than other progenitors, and they could phagocytoze apoptotic bodies. These data suggest that progenitors and stem cells share some characteristics of macrophages. This opens new perspectives on understanding stem cell phenotype and functionalities such as a putative role of stem cells in tissue remodeling by discarding dead cells but also their immunomodulation or fusion properties.

  14. Lymphocyte-to-monocyte ratio is associated with prognosis of diffuse large B-cell lymphoma: correlation with CD163 positive M2 type tumor-associated macrophages, not PD-1 positive tumor-infiltrating lymphocytes.

    PubMed

    Wang, Jingxuan; Gao, Kun; Lei, Wanting; Dong, Lina; Xuan, Qijia; Feng, Meiyan; Wang, Jinlu; Ye, Xiangnan; Jin, Tuan; Zhang, Zhongbai; Zhang, Qingyuan

    2017-01-17

    The research aims to examine the prognostic value of the lymphocyte-to-monocyte ratio (LMR), neutrophil-to- lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in diffuse large B-cell lymphoma (DLBCL). The relation of these hematologic indicators to poor antitumor immunity and prognosis must be investigated. Clinicopathologic data and survival information of 355 patients with DLBCL was retrospectively analyzed. Univariate analysis revealed that lower LMR (<2.71), higher NLR (≥2.81), CD163+ M2 tumor-associated macrophages (TAM) content ≥9.5% and programmed cell death 1 (PD-1)+ tumor-infiltrating lymphocytes (TILs) content < 4.5 cells per high power field(HPF) were significantly related to unfavorable overall survival (OS) and progression free survival (PFS). When considering the prognostic indexes of IPI, multivariate analysis confirmed that LMR of <2.71 and CD163+ M2 TAM content ≥9.5% significantly affected the prognosis of DLBCL. Spearman correlation test showed LMR was negatively correlated with CD163+ M2 TAM content. However, there were no correlation was found between LMR and PD-1+ TIL as well as between NLR and PD-1+ TIL content. These results indicated that decreased LMR lead to a weak anti-tumor immunity and could be used as a bad prognosis biomarker of DLBCL.

  15. Human serine protease HTRA1 positively regulates osteogenesis of human bone marrow-derived mesenchymal stem cells and mineralization of differentiating bone-forming cells through the modulation of extracellular matrix protein.

    PubMed

    Tiaden, André N; Breiden, Maike; Mirsaidi, Ali; Weber, Fabienne A; Bahrenberg, Gregor; Glanz, Stephan; Cinelli, Paolo; Ehrmann, Michael; Richards, Peter J

    2012-10-01

    Mammalian high-temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone-forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow-derived MSCs (hBMSCs), mouse adipose-derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin-binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating bone-forming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix.

  16. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed

    Colarusso, Pina; Spring, Kenneth R

    2002-02-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane.

  17. Reticulated lipid probe fluorescence reveals MDCK cell apical membrane topography.

    PubMed Central

    Colarusso, Pina; Spring, Kenneth R

    2002-01-01

    High spatial resolution confocal microscopy of young MDCK cells stained with the lipophilic probe 1,1'-dihexadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiIC(16)) revealed a reticulated fluorescence pattern on the apical membrane. DiIC(16) was delivered as crystals to live cells to minimize possible solvent perturbations of the membrane lipids. The ratio of the integrated fluorescence intensities in the bright versus dim regions was 1.6 +/- 0.1 (n = 13). Deconvolved images of the cells were consistent with exclusive plasma membrane staining. Multi-spectral and fluorescence anisotropy microscopy did not reveal differences between bright and dim regions. Bright regions coincided with microvilli and microridges observed by differential interference contrast microscopy and were stable for several minutes. Fluorescence recovery after photobleaching yielded similar diffusion coefficients (pooled D = 1.5 +/- 0.6 x 10(-9) cm(2)/s, n = 40) for both bright and dim regions. Line fluorescence recovery after photobleaching showed that the reticulated pattern was maintained as the fluorescence recovered in the bleached areas. Cytochalasin D did not affect the staining pattern, but the pattern was eliminated by cholesterol depletion with methyl-beta-cyclodextrin. We conclude that the reticulated fluorescence pattern was caused by increased optical path lengths through the microvilli and microridges compared with the flat areas on the apical membrane. PMID:11806917

  18. Revealing Transcriptome Landscape of Mouse Spermatogonial Cells by Tiling Microarray

    PubMed Central

    Lee, Tin-Lap.; Rennert, Owen M.; Chan, Wai-Yee.

    2014-01-01

    Summary Spermatogenesis is a highly regulated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific events through tightly regulated gene expression programs. In the past decade the advent of microarray technologies has allowed functional genomic studies of male germ cell development, resulting in the identification of genes governing various processes. A major limitation with conventional gene expression microarray is that there is a bias from gene probe design. The gene probes for expression microarrays are usually represented by a small number probes located at the 3’ end of a transcirpt. Tiling microarrays eliminate such issue by interrogating the genome in an unbiased fashion through probes tiled for the entire genome. These arrays provide a higher genomic resolution and allow identification of novel transcripts. To reveal the complexity of the genomic landscape of developing male germ cells, we applied tiling microarray to evaluate the transcriptome in spermatogonial cells. Over 50% of the mouse and rat genome are expressed during testicular development. More than 47% of transcripts are uncharacterized. The results suggested the transcription machinery in spermaotogonial cells are more complex than previously envisioned. PMID:22144238

  19. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    PubMed Central

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A.; Goren, Alon; Weitz, David A.; Bernstein, Bradley E.

    2015-01-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells, and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell is sparse, comprising on the order of 1000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of sub-populations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone. PMID:26458175

  20. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.

    PubMed

    Rotem, Assaf; Ram, Oren; Shoresh, Noam; Sperling, Ralph A; Goren, Alon; Weitz, David A; Bernstein, Bradley E

    2015-11-01

    Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.

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

    PubMed

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

    2017-03-29

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

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

    PubMed

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

    2013-02-11

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

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

    PubMed Central

    Chen, Weitao; Nie, Qing; Yi, Tau-Mu; Chou, Ching-Shan

    2016-01-01

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

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

    PubMed

    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.

  5. Cell Cycle and Cell Size Dependent Gene Expression Reveals Distinct Subpopulations at Single-Cell Level

    PubMed Central

    Dolatabadi, Soheila; Candia, Julián; Akrap, Nina; Vannas, Christoffer; Tesan Tomic, Tajana; Losert, Wolfgang; Landberg, Göran; Åman, Pierre; Ståhlberg, Anders

    2017-01-01

    Cell proliferation includes a series of events that is tightly regulated by several checkpoints and layers of control mechanisms. Most studies have been performed on large cell populations, but detailed understanding of cell dynamics and heterogeneity requires single-cell analysis. Here, we used quantitative real-time PCR, profiling the expression of 93 genes in single-cells from three different cell lines. Individual unsynchronized cells from three different cell lines were collected in different cell cycle phases (G0/G1 – S – G2/M) with variable cell sizes. We found that the total transcript level per cell and the expression of most individual genes correlated with progression through the cell cycle, but not with cell size. By applying the random forests algorithm, a supervised machine learning approach, we show how a multi-gene signature that classifies individual cells into their correct cell cycle phase and cell size can be generated. To identify the most predictive genes we used a variable selection strategy. Detailed analysis of cell cycle predictive genes allowed us to define subpopulations with distinct gene expression profiles and to calculate a cell cycle index that illustrates the transition of cells between cell cycle phases. In conclusion, we provide useful experimental approaches and bioinformatics to identify informative and predictive genes at the single-cell level, which opens up new means to describe and understand cell proliferation and subpopulation dynamics. PMID:28179914

  6. Revealing the structural and functional diversity of plant cell walls.

    PubMed

    Knox, J Paul

    2008-06-01

    The extensive knowledge of the chemistry of isolated cell wall polymers, and that relating to the identification and partial annotation of gene families involved in their synthesis and modification, is not yet matched by a sophisticated understanding of the occurrence of the polymers within cell walls of the diverse cell types within a growing organ. Currently, the main sets of tools that are used to determine cell-type-specific configurations of cell wall polymers and aspects of cell wall microstructures are antibodies, carbohydrate-binding modules (CBMs) and microspectroscopies. As these tools are applied we see that cell wall polymers are extensively developmentally regulated and that there is a range of structurally distinct primary and secondary cell walls within organs and across species. The challenge now is to document cell wall structures in relation to diverse cell biological events and to integrate this knowledge with the emerging understanding of polymer functions.

  7. Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

    PubMed Central

    Cai, Yunfei; Xenias, Harry; Spielman, Ingrid; Shneidman, Anna V.; David, Lawrence A.; Döbereiner, Hans-Günther; Wiggins, Chris H.; Sheetz, Michael P.

    2008-01-01

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

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. PMID:26349809

  9. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment

    PubMed Central

    van Wolfswinkel, Josien C.; Wagner, Daniel E.; Reddien, Peter W.

    2014-01-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments. PMID:25017721

  10. Metabolic Differences in Microbial Cell Populations Revealed by Nanophotonic Ionization

    SciTech Connect

    Walker, Bennett; Antonakos, Cory; Retterer, Scott T; Vertes, Akos

    2013-01-01

    ellular differences are linked to cell differentiation, the proliferation of cancer and to the development of drug resistance in microbial infections. Due to sensitivity limitations, however, large- scale metabolic analysis at the single cell level is only available for cells significantly larger in volume than Saccharomyces cerevisiae (~30 fL). Here we demonstrate that by a nanophotonic ionization platform and mass spectrometry, over one hundred up to 108 metabolites, or up to 18% of the known S. cerevisiae metabolome, can be identified in very small cell populations (n < 100). Under ideal conditions, r Relative quantitation of up to 4% of the metabolites is achieved at the single cell level.

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

    PubMed Central

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

    2017-01-01

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

  12. Advances in cell surface glycoengineering reveal biological function.

    PubMed

    Nischan, Nicole; Kohler, Jennifer J

    2016-08-01

    Cell surface glycans are critical mediators of cell-cell, cell-ligand, and cell-pathogen interactions. By controlling the set of glycans displayed on the surface of a cell, it is possible to gain insight into the biological functions of glycans. Moreover, control of glycan expression can be used to direct cellular behavior. While genetic approaches to manipulate glycosyltransferase gene expression are available, their utility in glycan engineering has limitations due to the combinatorial nature of glycan biosynthesis and the functional redundancy of glycosyltransferase genes. Biochemical and chemical strategies offer valuable complements to these genetic approaches, notably by enabling introduction of unnatural functionalities, such as fluorophores, into cell surface glycans. Here, we describe some of the most recent developments in glycoengineering of cell surfaces, with an emphasis on strategies that employ novel chemical reagents. We highlight key examples of how these advances in cell surface glycan engineering enable study of cell surface glycans and their function. Exciting new technologies include synthetic lipid-glycans, new chemical reporters for metabolic oligosaccharide engineering to allow tandem and in vivo labeling of glycans, improved chemical and enzymatic methods for glycoproteomics, and metabolic glycosyltransferase inhibitors. Many chemical and biochemical reagents for glycan engineering are commercially available, facilitating their adoption by the biological community.

  13. Cell dipole behaviour revealed by ECM sub-cellular geometry

    NASA Astrophysics Data System (ADS)

    Mandal, Kalpana; Wang, Irène; Vitiello, Elisa; Orellana, Laura Andreina Chacòn; Balland, Martial

    2014-12-01

    Cells sense and respond to their mechanical environment by exerting forces on their surroundings. The way forces are modulated by extra-cellular matrix (ECM) properties plays a key role in tissue homoeostasis. Using highly resolved micropatterns that constrain cells into the same square envelope but vary the adhesive geometry, here we investigate how the adhesive micro-environment affects the architecture of actin cytoskeleton and the orientation of traction forces. Our data demonstrate that local adhesive changes can trigger orientational ordering of stress fibres throughout the cell, suggesting that cells are capable of integrating information on ECM geometry at the whole-cell level. Finally, we show that cells tend to generate highly polarized force pattern, that is, unidirectional pinching, in response to adequate adhesive conditions. Hence, the geometry of adhesive environment can induce cellular orientation, a process which may have significant implications for the formation and mechanical properties of tissues.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  16. Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

    PubMed Central

    Mulhall, Hayley J.; Marchenko, Steve A.; Hoettges, Kai F.; Estrada, Laura C.; Lee, Abraham P.; Hughes, Michael P.; Flanagan, Lisa A.

    2011-01-01

    Background Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. Methodology/Principal Findings We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. Conclusions/Significance We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors. PMID:21980464

  17. Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

    PubMed Central

    Brawley, Crista M.; Rock, Ronald S.

    2009-01-01

    Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments. PMID:19478066

  18. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells.

    PubMed

    Lawson, Devon A; Bhakta, Nirav R; Kessenbrock, Kai; Prummel, Karin D; Yu, Ying; Takai, Ken; Zhou, Alicia; Eyob, Henok; Balakrishnan, Sanjeev; Wang, Chih-Yang; Yaswen, Paul; Goga, Andrei; Werb, Zena

    2015-10-01

    Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated

  19. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

    PubMed Central

    Estacion, Mark; Schilling, William P

    2002-01-01

    Background Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. Results Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. Conclusions Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms. PMID:11825342

  20. Brief Report: Single-Cell Analysis Reveals Cell Division-Independent Emergence of Megakaryocytes From Phenotypic Hematopoietic Stem Cells.

    PubMed

    Roch, Aline; Trachsel, Vincent; Lutolf, Matthias P

    2015-10-01

    Despite increasingly stringent methods to isolate hematopoietic stem cells (HSCs), considerable heterogeneity remains in terms of their long-term self-renewal and differentiation potential. Recently, the existence of long-lived, self-renewing, myeloid-restricted progenitors in the phenotypically defined HSC compartment has been revealed, but these cells remain poorly characterized. Here, we used an in vitro single-cell analysis approach to track the fate of 330 long-term HSCs (LT-HSC; Lin- cKit+ Sca-1+ CD150+ CD48- CD34-) cultured for 5 days under serum-free basal conditions. Our analysis revealed a highly heterogeneous behavior with approximately 15% of all phenotypic LT-HSCs giving rise to megakaryocytes (Mk). Surprisingly, in 65% of these cases, Mk development occurred in the absence of cell division. This observation suggests that myeloid-restricted progenitors may not derive directly from LT-HSCs but instead could share an identical cell surface marker repertoire.

  1. Exosomal HIF1α supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes.

    PubMed

    Aga, M; Bentz, G L; Raffa, S; Torrisi, M R; Kondo, S; Wakisaka, N; Yoshizaki, T; Pagano, J S; Shackelford, J

    2014-09-11

    It has emerged recently that exosomes are potential carriers of pro-tumorigenic factors that participate in oncogenesis. However, whether oncogenic transcription factors are transduced by exosomes is unknown. Hypoxia-inducible factor-1α (HIF1α) transcriptionally regulates numerous key aspects of tumor development and progression by promoting a more aggressive tumor phenotype, characterized by increased proliferation and invasiveness coupled with neoangiogenesis. It has been shown that the principal oncoprotein of Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), drives oncogenic processes and tumor progression of the highly invasive EBV malignancy, nasopharyngeal carcinoma (NPC). We now demonstrate that endogenous HIF1α is detectable in exosomes and that LMP1 significantly increases levels of HIF1α in exosomes. HIF1 recovered from exosomes retains DNA-binding activity and is transcriptionally active in recipient cells after exosome uptake. We also show that treatment of EBV-negative cells with LMP1-exosomes increases migration and invasiveness of NP cell lines in functional assays, which correlates with the phenotype associated with epithelial-mesenchymal transition (EMT). In addition, we provide evidence that HIF1α itself participates in exosome-mediated pro-metastatic effects in recipient cells, as exosome-mediated delivery of active and inactive forms of HIF1α results in reciprocal changes in the expression of E- and N-cadherins associated with EMT. Further, immunohistochemical analysis of NPC tumor tissues revealed direct correlation between protein levels of LMP1 and of the endosome/exosome marker tetraspanin, CD63, which suggests an increase in exosome formation in this EBV-positive malignancy. We hypothesize that exosome-mediated transfer of functional pro-metastatic factors by LMP1-positive NPC cells to surrounding tumor cells promotes cancer progression.

  2. Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

    PubMed Central

    Huang, Yanmei; Ainsley, Joshua A.; Reijmers, Leon G.; Jackson, F. Rob

    2013-01-01

    Abstract Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity. PMID:24348200

  3. Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils.

    PubMed

    Kuckleburg, Christopher J; Tilkens, Sarah B; Santoso, Sentot; Newman, Peter J

    2012-03-01

    Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.

  4. Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

    PubMed Central

    Isensee, Jörg; Wenzel, Carsten; Buschow, Rene; Weissmann, Robert; Kuss, Andreas W.; Hucho, Tim

    2014-01-01

    Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p<0.05, fold-change>1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD2 activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD2 producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based

  5. Cell lineage tracing reveals a biliary origin of intrahepatic cholangiocarcinoma

    PubMed Central

    Guest, Rachel V; Boulter, Luke; Kendall, Timothy J; Minnis-Lyons, Sarah E; Walker, Robert; Wigmore, Stephen J; Sansom, Owen J; Forbes, Stuart J

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signalling within mature hepatocytes is able to induce the formation of tumours displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumour. Here we utilise a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labelled biliary lineage tumours in response to chronic injury. Consequent to this, up-regulation of native functional Notch signalling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human ICC. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of ICC. PMID:24310400

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

    DOE PAGES

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; ...

    2016-01-21

    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-resolutionmore » 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. We find 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. Lastly, these observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

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

    SciTech Connect

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Urban, Volker S.; Ohl, Michael

    2016-01-21

    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. We find 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. Lastly, these observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Metabolic profiling reveals key metabolic features of renal cell carcinoma.

    PubMed

    Catchpole, Gareth; Platzer, Alexander; Weikert, Cornelia; Kempkensteffen, Carsten; Johannsen, Manfred; Krause, Hans; Jung, Klaus; Miller, Kurt; Willmitzer, Lothar; Selbig, Joachim; Weikert, Steffen

    2011-01-01

    Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α-tocopherol, hippuric acid, myoinositol, fructose-1-phosphate and glucose-1-phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC.

  11. Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells

    PubMed Central

    Ene, Iuliana V.; Lohse, Matthew B.; Vladu, Adrian V.; Morschhäuser, Joachim; Johnson, Alexander D.

    2016-01-01

    ABSTRACT The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state. PMID:27879329

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

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

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

  13. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.

    PubMed

    Shalek, Alex K; Satija, Rahul; Shuga, Joe; Trombetta, John J; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S; Gaublomme, Jellert T; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P; Regev, Aviv

    2014-06-19

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a 'core' module of antiviral genes is expressed very early by a few 'precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced 'peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

  14. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation

    NASA Astrophysics Data System (ADS)

    Shalek, Alex K.; Satija, Rahul; Shuga, Joe; Trombetta, John J.; Gennert, Dave; Lu, Diana; Chen, Peilin; Gertner, Rona S.; Gaublomme, Jellert T.; Yosef, Nir; Schwartz, Schraga; Fowler, Brian; Weaver, Suzanne; Wang, Jing; Wang, Xiaohui; Ding, Ruihua; Raychowdhury, Raktima; Friedman, Nir; Hacohen, Nir; Park, Hongkun; May, Andrew P.; Regev, Aviv

    2014-06-01

    High-throughput single-cell transcriptomics offers an unbiased approach for understanding the extent, basis and function of gene expression variation between seemingly identical cells. Here we sequence single-cell RNA-seq libraries prepared from over 1,700 primary mouse bone-marrow-derived dendritic cells spanning several experimental conditions. We find substantial variation between identically stimulated dendritic cells, in both the fraction of cells detectably expressing a given messenger RNA and the transcript's level within expressing cells. Distinct gene modules are characterized by different temporal heterogeneity profiles. In particular, a `core' module of antiviral genes is expressed very early by a few `precocious' cells in response to uniform stimulation with a pathogenic component, but is later activated in all cells. By stimulating cells individually in sealed microfluidic chambers, analysing dendritic cells from knockout mice, and modulating secretion and extracellular signalling, we show that this response is coordinated by interferon-mediated paracrine signalling from these precocious cells. Notably, preventing cell-to-cell communication also substantially reduces variability between cells in the expression of an early-induced `peaked' inflammatory module, suggesting that paracrine signalling additionally represses part of the inflammatory program. Our study highlights the importance of cell-to-cell communication in controlling cellular heterogeneity and reveals general strategies that multicellular populations can use to establish complex dynamic responses.

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

    PubMed

    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.

  16. Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

    PubMed

    Shin, Jaehoon; Berg, Daniel A; Zhu, Yunhua; Shin, Joseph Y; Song, Juan; Bonaguidi, Michael A; Enikolopov, Grigori; Nauen, David W; Christian, Kimberly M; Ming, Guo-li; Song, Hongjun

    2015-09-03

    Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

  17. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  18. Cell-material interactions revealed via material techniques of surface patterning.

    PubMed

    Yao, Xiang; Peng, Rong; Ding, Jiandong

    2013-10-04

    Cell-material interactions constitute a key fundamental topic in biomaterials study. Various cell cues and matrix cues as well as soluble factors regulate cell behaviors on materials. These factors are coupled with each other as usual, and thus it is very difficult to unambiguously elucidate the role of each regulator. The recently developed material techniques of surface patterning afford unique ways to reveal the underlying science. This paper reviews the pertinent material techniques to fabricate patterns of microscale and nanoscale resolutions, and corresponding cell studies. Some issues are emphasized, such as cell localization on patterned surfaces of chemical contrast, and effects of cell shape, cell size, cell-cell contact, and seeding density on differentiation of stem cells. Material cues to regulate cell adhesion, cell differentiation and other cell events are further summed up. Effects of some physical properties, such as surface topography and matrix stiffness, on cell behaviors are also discussed; nanoscaled features of substrate surfaces to regulate cell fate are summarized as well. The pertinent work sheds new insight into the cell-material interactions, and is stimulating for biomaterial design in regenerative medicine, tissue engineering, and high-throughput detection, diagnosis, and drug screening.

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

    PubMed

    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.

  20. Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness.

    PubMed

    Monds, Russell D; Lee, Timothy K; Colavin, Alexandre; Ursell, Tristan; Quan, Selwyn; Cooper, Tim F; Huang, Kerwyn Casey

    2014-11-20

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

  1. Variation in Carbohydrates between Cancer and Normal Cell Membranes Revealed by Super‐Resolution Fluorescence Imaging

    PubMed Central

    Chen, Junling; Liu, Tianzhou; Gao, Jing; Gao, Lan; Zhou, Lulu; Cai, Mingjun; Shi, Yan; Xiong, Wenyong; Jiang, Junguang

    2016-01-01

    Carbohydrate alterations on cell membranes are associated with various cancer processes, including tumorigenesis, malignant transformation, and tumor dissemination. However, variations in the distributions of cancer‐associated carbohydrates are unclear at the molecular level. Herein, direct stochastic optical reconstruction microscopy is used to reveal that seven major types of carbohydrates tended to form obvious clusters on cancer cell membranes compared with normal cell membranes (both cultured and primary cells), and most types of carbohydrates present a similar distributed characteristic on various cancer cells (e.g., HeLa and Os‐Rc‐2 cells). Significantly, sialic acid is found to distribute in larger‐sized clusters with a higher cluster coverage percentage on various cancer cells than normal cells. These findings on the aberrant distributions of cancer‐associated carbohydrates can potentially serve as novel diagnostic and therapeutic targets, as well as making a contribution to clarify how abnormal glycosylations of membrane glycoconjugates participate in tumorigenesis and metastasis. PMID:27981014

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

    PubMed Central

    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

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

    PubMed

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

    2015-03-01

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

  4. Continuous single cell imaging reveals sequential steps of plasmacytoid dendritic cell development from common dendritic cell progenitors

    PubMed Central

    Dursun, Ezgi; Endele, Max; Musumeci, Andrea; Failmezger, Henrik; Wang, Shu-Hung; Tresch, Achim; Schroeder, Timm; Krug, Anne B.

    2016-01-01

    Functionally distinct plasmacytoid and conventional dendritic cells (pDC and cDC) shape innate and adaptive immunity. They are derived from common dendritic cell progenitors (CDPs) in the murine bone marrow, which give rise to CD11c+ MHCII− precursors with early commitment to DC subpopulations. In this study, we dissect pDC development from CDP into an ordered sequence of differentiation events by monitoring the expression of CD11c, MHC class II, Siglec H and CCR9 in CDP cultures by continuous single cell imaging and tracking. Analysis of CDP genealogies revealed a stepwise differentiation of CDPs into pDCs in a part of the CDP colonies. This developmental pathway involved an early CD11c+ SiglecH− pre-DC stage and a Siglec H+ CCR9low precursor stage, which was followed rapidly by upregulation of CCR9 indicating final pDC differentiation. In the majority of the remaining CDP pedigrees however the Siglec H+ CCR9low precursor state was maintained for several generations. Thus, although a fraction of CDPs transits through precursor stages rapidly to give rise to a first wave of pDCs, the majority of CDP progeny differentiate more slowly and give rise to longer lived precursor cells which are poised to differentiate on demand. PMID:27892478

  5. A simple engineered platform reveals different modes of tumor-microenvironmental cell interaction

    PubMed Central

    Zhang, Chentian; Shenk, Elizabeth M; Blaha, Laura C; Ryu, Byungwoo; Alani, Rhoda M; Cabodi, Mario; Wong, Joyce Y

    2016-01-01

    How metastatic cancer lesions survive and grow in secondary locations is not fully understood. There is a growing appreciation for the importance of tumor components, i.e. microenvironmental cells, in this process. Here, we used a simple microfabricated dual cell culture platform with a 500 μm gap to assess interactions between two different metastatic melanoma cell lines (1205Lu isolated from a lung lesion established through a mouse xenograft; and WM852 derived from a stage III metastatic lesion of skin) and microenvironmental cells derived from either skin (fibroblasts), lung (epithelial cells) or liver (hepatocytes). We observed differential bi-directional migration between microenvironmental cells and melanoma, depending on the melanoma cell line. Lung epithelial cells and skin fibroblasts, but not hepatocytes, stimulated higher 1205Lu migration than without microenvironmental cells; in the opposite direction, 1205Lu cells induced hepatocytes to migrate, but had no effect on skin fibroblasts and slightly inhibited lung epithelial cells. In contrast, none of the microenvironments had a significant effect on WM852; in this case, skin fibroblasts and hepatocytes—but not lung epithelial cells—exhibited directed migration toward WM852. These observations reveal significant effects a given microenvironmental cell line has on the two different melanoma lines, as well as how melanoma effects different microenvironmental cell lines. Our simple platform thus has potential to provide complex insights into different strategies used by cancerous cells to survive in and colonize metastatic sites. PMID:26716792

  6. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  7. Lineage tracing quantification reveals symmetric stem cell division in Drosophila male germline stem cells.

    PubMed

    Salzmann, Viktoria; Inaba, Mayu; Cheng, Jun; Yamashita, Yukiko M

    2013-12-01

    In the homeostatic state, adult stem cells divide either symmetrically to increase the stem cell number to compensate stem cell loss, or asymmetrically to maintain the population while producing differentiated cells. We have investigated the mode of stem cell division in the testes of Drosophila melanogaster by lineage tracing and confirm the presence of symmetric stem cell division in this system. We found that the rate of symmetric division is limited to 1-2% of total germline stem cell (GSC) divisions, but it increases with expression of a cell adhesion molecule, E-cadherin, or a regulator of the actin cytoskeleton, Moesin, which may modulate adhesiveness of germ cells to the stem cell niche. Our results indicate that the decision regarding asymmetric vs. symmetric division is a dynamically regulated process that contributes to tissue homeostasis, responding to the needs of the tissue.

  8. Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic Cell-mediated Antigen Presentation

    PubMed Central

    Fehlings, Michael; Drobbe, Lea; Beigier-Bompadre, Macarena; Viveros, Pablo Renner; Moos, Verena; Schneider, Thomas; Meyer, Thomas F.; Aebischer, Toni; Ignatius, Ralf

    2016-01-01

    Direct effects of Helicobacter pylori (H. pylori) on human CD4+ T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1+ monocyte-derived dendritic cells were exposed to H. pylori and pulsed with the antigen 85B from Mycobacterium tuberculosis (M. tuberculosis). Interleukin-2 (IL-2) secretion by AG85Baa97-112-specific hybridoma cells was then evaluated as an integral reporter of cognate antigen presentation. This methodology enabled revealing of interference of H. pylori with the antigen-presenting capacity of human dendritic cells. PMID:27980859

  9. YY1 positively regulates human UBIAD1 expression

    SciTech Connect

    Funahashi, Nobuaki; Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natumi; Watanabe, Masato; Suhara, Yoshitomo; Okano, Toshio

    2015-05-01

    Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K{sub 1}) and a series of bacterial menaquionones (MK-n; vitamin K{sub 2}). Menadione (vitamin K{sub 3}) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5′ rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter. - Highlights: • We cloned the human UBIAD1 promoter. • The functional importance of the YY1 motif was identified in the UBIAD1 promoter. • YY1 binds the UBIAD1 promoter in vitro and in vivo. • Knockdown of YY1 significantly decreased UBIAD1 expression. • YY1 up-regulates UBIAD1 conversion activity through the UBIAD1

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

    PubMed

    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.

  11. Single-cell analysis reveals lineage segregation in early post-implantation mouse embryos.

    PubMed

    Wen, Jing; Zeng, Yanwu; Fang, Zhuoqing; Gu, Junjie; Ge, Laixiang; Tang, Fan; Qu, Zepeng; Hu, Jing; Cui, Yaru; Zhang, Kunshan; Wang, Junbang; Li, Siguang; Sun, Yi; Jin, Ying

    2017-03-15

    The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. Especially, the individual definitive endoderm (DE) cells have not been characterized in vivo. Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (Fgf) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the pre-mesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6. Analysis of single-cell RNA-seq data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.

  12. Single-cell messenger RNA sequencing reveals rare intestinal cell types.

    PubMed

    Grün, Dominic; Lyubimova, Anna; Kester, Lennart; Wiebrands, Kay; Basak, Onur; Sasaki, Nobuo; Clevers, Hans; van Oudenaarden, Alexander

    2015-09-10

    Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5

  13. Data on in vivo phenotypes of GFRα1-positive spermatogonia stimulated by interstitial GDNF signals in mouse testes.

    PubMed

    Uchida, Aya; Kanai, Yoshiakira

    2016-09-01

    This article contains the data related to the research article "in vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay" (Uchida et al., 2016) [1]. A novel transplantation assay of growth factor-soaked beads into the mammalian testicular interstitium was developed, in order to examine the effects of various soluble factors on in vivo dynamics of the spermatogonia including spermatogonial stem cells (SSC). Here we provide the image data of GFRα1-positive stem/progenitor spermatogonia in mouse seminiferous tubules near the beads soaked in GDNF (glial cell-derived neurotrophic factor), one of the SSC niche factors. The data provide various phenotypes of GFRα1-positive spermatogonia induced by bead-derived GDNF signals, which are useful to understand the active state of GFRα1-positive stem/progenitor spermatogonia in vivo.

  14. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

    PubMed

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-05-05

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.

  15. A Quorum-Sensing Factor in Vegetative Dictyostelium Discoideum Cells Revealed by Quantitative Migration Analysis

    PubMed Central

    Golé, Laurent; Rivière, Charlotte; Hayakawa, Yoshinori; Rieu, Jean-Paul

    2011-01-01

    Background Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS). In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. Methods and Findings To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (), but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF) on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. Conclusion This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase. PMID:22073217

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of Ferroptosis

    PubMed Central

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J.; Brown, Lewis M.; Valenzuela, Carlos A.; Wolpaw, Adam J.

    2016-01-01

    Apoptosis is known as programmed cell death. Some non-apoptotic cell death is increasingly recognized as genetically controlled, or ‘regulated’. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. Here, we surveyed the landscape of pharmacologically-accessible cell death mechanisms. Of 56 caspase-independent lethal compounds, modulatory profiling revealed ten inducing three types of regulated non-apoptotic cell death. Lead optimization of one of the ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis occurs when the lipid repair enzyme GPX4 is inhibited. We found that FIN56 promotes degradation of GPX4. We performed chemoproteomics to reveal that FIN56 also binds to and activates squalene synthase, an enzyme involved in the cholesterol synthesis, in a manner independent of GPX4 degradation. These discoveries reveal that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes. PMID:27159577

  18. Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium

    PubMed Central

    Watson, Julie K.; Rulands, Steffen; Wilkinson, Adam C.; Wuidart, Aline; Ousset, Marielle; Van Keymeulen, Alexandra; Göttgens, Berthold; Blanpain, Cédric; Simons, Benjamin D.; Rawlins, Emma L.

    2015-01-01

    Summary Epithelial lineages have been studied at cellular resolution in multiple organs that turn over rapidly. However, many epithelia, including those of the lung, liver, pancreas, and prostate, turn over slowly and may be regulated differently. We investigated the mouse tracheal epithelial lineage at homeostasis by using long-term clonal analysis and mathematical modeling. This pseudostratified epithelium contains basal cells and secretory and multiciliated luminal cells. Our analysis revealed that basal cells are heterogeneous, comprising approximately equal numbers of multipotent stem cells and committed precursors, which persist in the basal layer for 11 days before differentiating to luminal fate. We confirmed the molecular and functional differences within the basal population by using single-cell qRT-PCR and further lineage labeling. Additionally, we show that self-renewal of short-lived secretory cells is a feature of homeostasis. We have thus revealed early luminal commitment of cells that are morphologically indistinguishable from stem cells. PMID:26119728

  19. Branching process deconvolution algorithm reveals a detailed cell-cycle transcription program.

    PubMed

    Guo, Xin; Bernard, Allister; Orlando, David A; Haase, Steven B; Hartemink, Alexander J

    2013-03-05

    Due to cell-to-cell variability and asymmetric cell division, cells in a synchronized population lose synchrony over time. As a result, time-series measurements from synchronized cell populations do not reflect the underlying dynamics of cell-cycle processes. Here, we present a branching process deconvolution algorithm that learns a more accurate view of dynamic cell-cycle processes, free from the convolution effects associated with imperfect cell synchronization. Through wavelet-basis regularization, our method sharpens signal without sharpening noise and can remarkably increase both the dynamic range and the temporal resolution of time-series data. Although applicable to any such data, we demonstrate the utility of our method by applying it to a recent cell-cycle transcription time course in the eukaryote Saccharomyces cerevisiae. Our method more sensitively detects cell-cycle-regulated transcription and reveals subtle timing differences that are masked in the original population measurements. Our algorithm also explicitly learns distinct transcription programs for mother and daughter cells, enabling us to identify 82 genes transcribed almost entirely in early G1 in a daughter-specific manner.

  20. Transcriptional profile of TB antigen-specific T cells reveals novel multifunctional features1

    PubMed Central

    Arlehamn, Cecilia Lindestam; Seumois, Gregory; Gerasimova, Anna; Huang, Charlie; Fu, Zheng; Yue, Xiaojing; Sette, Alessandro; Vijayanand, Pandurangan; Peters, Bjoern

    2014-01-01

    In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4+ T cells as important contributors. Here we show that TB-specific CD4+ T cells have a characteristic chemokine expression signature (CCR6+CXCR3+CCR4−), and that the overall number of these cells is significantly increased in LTBI donors compared to healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6+CXCR3+CCR4− cells and find significant differences to conventional Th1, Th17 and Th2 cells, but no major changes between healthy and LTBI donors. CCR6+CXCR3+CCR4− cells display linage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6+CXCR3+CCR4− cells reveals characteristics important for controlling latent TB infections. PMID:25092889

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

    SciTech Connect

    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.

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

    PubMed

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

    2012-04-01

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

  3. Selective Localization of Shanks to VGLUT1-Positive Excitatory Synapses in the Mouse Hippocampus

    PubMed Central

    Heise, Christopher; Schroeder, Jan C.; Schoen, Michael; Halbedl, Sonja; Reim, Dominik; Woelfle, Sarah; Kreutz, Michael R.; Schmeisser, Michael J.; Boeckers, Tobias M.

    2016-01-01

    Members of the Shank family of multidomain proteins (Shank1, Shank2, and Shank3) are core components of the postsynaptic density (PSD) of excitatory synapses. At synaptic sites Shanks serve as scaffolding molecules that cluster neurotransmitter receptors as well as cell adhesion molecules attaching them to the actin cytoskeleton. In this study we investigated the synapse specific localization of Shank1-3 and focused on well-defined synaptic contacts within the hippocampal formation. We found that all three family members are present only at VGLUT1-positive synapses, which is particularly visible at mossy fiber contacts. No costaining was found at VGLUT2-positive contacts indicating that the molecular organization of VGLUT2-associated PSDs diverges from classical VGLUT1-positive excitatory contacts in the hippocampus. In light of SHANK mutations in neuropsychiatric disorders, this study indicates which glutamatergic networks within the hippocampus will be primarily affected by shankopathies. PMID:27199660

  4. Heterogeneity of neural progenitor cells revealed by enhancers in the nestin gene

    PubMed Central

    Yaworsky, Paul J.; Kappen, Claudia

    2014-01-01

    Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory elements. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations. PMID:9917366

  5. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

    PubMed

    Deng, Qiaolin; Ramsköld, Daniel; Reinius, Björn; Sandberg, Rickard

    2014-01-10

    Expression from both alleles is generally observed in analyses of diploid cell populations, but studies addressing allelic expression patterns genome-wide in single cells are lacking. Here, we present global analyses of allelic expression across individual cells of mouse preimplantation embryos of mixed background (CAST/EiJ × C57BL/6J). We discovered abundant (12 to 24%) monoallelic expression of autosomal genes and that expression of the two alleles occurs independently. The monoallelic expression appeared random and dynamic because there was considerable variation among closely related embryonic cells. Similar patterns of monoallelic expression were observed in mature cells. Our allelic expression analysis also demonstrates the de novo inactivation of the paternal X chromosome. We conclude that independent and stochastic allelic transcription generates abundant random monoallelic expression in the mammalian cell.

  6. Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells

    PubMed Central

    Lee, Timothy K.; Meng, Kevin; Shi, Handuo; Huang, Kerwyn Casey

    2016-01-01

    The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis. PMID:27774981

  7. Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen Presenting Cells, Critical for T cell Immunity

    PubMed Central

    Broz, Miranda; Binnewies, Mikhail; Boldajipour, Bijan; Nelson, Amanda; Pollock, Joshua; Erle, David; Barczak, Andrea; Rosenblum, Michael; Daud, Adil; Barber, Diane; Amigorena, Sebastian; van’t Veer, Laura J.; Sperling, Anne; Wolf, Denise; Krummel, Matthew F.

    2014-01-01

    SUMMARY It is well understood that antigen-presenting cells (APC) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic-cell (DC) populations as distinct from macrophage populations. Within these, CD103+ DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent upon IRF8, Zbtb46 and Batf3 transcription factors and generated by GM-CSF and Flt3L cytokines. Regressing tumors have higher proportions of these cells, T-cell dependent immune clearance relies upon them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types. PMID:25446897

  8. Whole population cell analysis of a landmark-rich mammalian epithelium reveals multiple elongation mechanisms

    PubMed Central

    Economou, Andrew D.; Brock, Lara J.; Cobourne, Martyn T.; Green, Jeremy B. A.

    2013-01-01

    Tissue elongation is a fundamental component of developing and regenerating systems. Although localised proliferation is an important mechanism for tissue elongation, potentially important contributions of other elongation mechanisms, specifically cell shape change, orientated cell division and cell rearrangement, are rarely considered or quantified, particularly in mammalian systems. Their quantification, together with proliferation, provides a rigorous framework for the analysis of elongation. The mammalian palatal epithelium is a landmark-rich tissue, marked by regularly spaced ridges (rugae), making it an excellent model in which to analyse the contributions of cellular processes to directional tissue growth. We captured confocal stacks of entire fixed mouse palate epithelia throughout the mid-gestation growth period, labelled with membrane, nuclear and cell proliferation markers and segmented all cells (up to ∼20,000 per palate), allowing the quantification of cell shape and proliferation. Using the rugae as landmarks, these measures revealed that the so-called growth zone is a region of proliferation that is intermittently elevated at ruga initiation. The distribution of oriented cell division suggests that it is not a driver of tissue elongation, whereas cell shape analysis revealed that both elongation of cells leaving the growth zone and apico-basal cell rearrangements do contribute significantly to directional growth. Quantitative comparison of elongation processes indicated that proliferation contributes most to elongation at the growth zone, but cell shape change and rearrangement contribute as much as 40% of total elongation. We have demonstrated the utility of an approach to analysing the cellular mechanisms underlying tissue elongation in mammalian tissues. It should be broadly applied to higher-resolution analysis of links between genotypes and malformation phenotypes. PMID:24173805

  9. Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons

    PubMed Central

    Földy, Csaba; Darmanis, Spyros; Aoto, Jason; Malenka, Robert C.; Quake, Stephen R.; Südhof, Thomas C.

    2016-01-01

    In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity. PMID:27531958

  10. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

    PubMed

    Agirre, Xabier; Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C; Beekman, Renée; Rodríguez-Madoz, Juan R; San José-Enériz, Edurne; Fang, Fang; Gutiérrez, Norma C; García-Verdugo, José M; Robson, Michael I; Schirmer, Eric C; Guruceaga, Elisabeth; Martens, Joost H A; Gut, Marta; Calasanz, Maria J; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F San; Melnick, Ari; Stunnenberg, Hendrik G; Gut, Ivo G; Prosper, Felipe; Martín-Subero, José I

    2015-04-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

  11. Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers

    PubMed Central

    Castellano, Giancarlo; Pascual, Marien; Heath, Simon; Kulis, Marta; Segura, Victor; Bergmann, Anke; Esteve, Anna; Merkel, Angelika; Raineri, Emanuele; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Russiñol, Nuria; Queirós, Ana C.; Beekman, Renée; Rodríguez-Madoz, Juan R.; José-Enériz, Edurne San; Fang, Fang; Gutiérrez, Norma C.; García-Verdugo, José M.; Robson, Michael I.; Schirmer, Eric C.; Guruceaga, Elisabeth; Martens, Joost H.A.; Gut, Marta; Calasanz, Maria J.; Flicek, Paul; Siebert, Reiner; Campo, Elías; Miguel, Jesús F. San; Melnick, Ari; Stunnenberg, Hendrik G.; Gut, Ivo G.

    2015-01-01

    While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM. PMID:25644835

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

    PubMed

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

    2016-06-03

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

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

    PubMed Central

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

    2011-01-01

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

  14. Genetically induced cell death in bulge stem cells reveals their redundancy for hair and epidermal regeneration.

    PubMed

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

    2015-03-01

    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 Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent.

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

    PubMed Central

    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

  16. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibañez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  17. Single-Cell mRNA Profiling Reveals Cell-Type Specific Expression of Neurexin Isoforms

    PubMed Central

    Fuccillo, Marc V.; Földy, Csaba; Gökce, Özgün; Rothwell, Patrick E.; Sun, Gordon L.; Malenka, Robert C.; Südhof, Thomas C.

    2016-01-01

    Summary Neurexins are considered central organizers of synapse architecture that are implicated in neuropsychiatric disorders. Expression of neurexins in hundreds of alternatively spliced isoforms suggested that individual neurons might exhibit a cell type-specific neurexin expression pattern (a neurexin code). To test this hypothesis, we quantified the single-cell levels of neurexin isoforms and other trans-synaptic cell-adhesion molecules by microfluidics-based RT-PCR. We show that the neurexin repertoire displays pronounced cell-type specificity that is remarkably consistent within each type of neuron. Furthermore, we uncovered region-specific regulation of neurexin transcription and splice-site usage. Finally, we demonstrate that the transcriptional profiles of neurexins can be altered in an experience-dependent fashion by exposure to a drug of abuse. Our data provide evidence of cell type-specific expression patterns of multiple neurexins at the single-cell level, and suggest that expression of synaptic cell-adhesion molecules overlaps with other key features of cellular identity and diversity. PMID:26182417

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

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

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

    PubMed Central

    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

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

    PubMed

    Sergé, Arnauld

    2016-01-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed Central

    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

  3. Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

    PubMed Central

    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.

    2015-01-01

    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. PMID:25667316

  4. Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

    SciTech Connect

    Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

    2009-02-01

    The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.

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

    PubMed

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

    2017-02-15

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

  6. Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

    PubMed Central

    Cheeseman, Bevan L.; Zhang, Dongcheng; Binder, Benjamin J.; Newgreen, Donald F.; Landman, Kerry A.

    2014-01-01

    Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS. PMID:24501272

  7. Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

    PubMed Central

    Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

    2014-01-01

    Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  9. Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

    PubMed

    Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

    2017-03-03

    Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10(5) to 1 × 10(6 )cells/mm(3). Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.

  10. Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids

    PubMed Central

    Schmitz, Alexander; Fischer, Sabine C.; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H. K.

    2017-01-01

    Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid’s size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 105 to 1 × 106 cells/mm3. Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture. PMID:28255161

  11. Identification of essential Alphaproteobacterial genes reveals operational variability in conserved developmental and cell cycle systems

    PubMed Central

    Curtis, Patrick D.; Brun, Yves V.

    2014-01-01

    Summary The cell cycle of Caulobacter crescentus is controlled by a complex signaling network that coordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism’s different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism’s essential gene pool is specific to that organism. PMID:24975755

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

    PubMed Central

    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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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.

  14. An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

    PubMed Central

    Schütte, Judith; Wang, Huange; Antoniou, Stella; Jarratt, Andrew; Wilson, Nicola K; Riepsaame, Joey; Calero-Nieto, Fernando J; Moignard, Victoria; Basilico, Silvia; Kinston, Sarah J; Hannah, Rebecca L; Chan, Mun Chiang; Nürnberg, Sylvia T; Ouwehand, Willem H; Bonzanni, Nicola; de Bruijn, Marella FTR; Göttgens, Berthold

    2016-01-01

    Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. DOI: http://dx.doi.org/10.7554/eLife.11469.001 PMID:26901438

  15. Electron and carbon balances in microbial fuel cells reveal temporary bacterial storage behavior during electricity generation.

    PubMed

    Freguia, Stefano; Rabaey, Korneel; Yuan, Zhiguo; Keller, Jürg

    2007-04-15

    Microbial fuel cells (MFCs) are emerging as a novel technology with a great potential to reduce the costs of wastewater treatment. Their most studied application is organic carbon removal. One of the parameters commonly used to quantify the performance of these cells is the Coulombic efficiency, i.e., the electron recovery as electricity from the removed substrate. However, the "inefficiencies" of the process have never been fully identified. This study presents a method that uses the combination of electrochemical monitoring, chemical analysis, and a titration and off-gas analysis (TOGA) sensor to identify and quantify the sources of electron loss. The method was used successfully to close electron, carbon, and proton balances in acetate and glucose fed microbial fuel cells. The method revealed that in the case that a substrate is loaded as pulses carbon is stored inside the cells during initial high substrate conditions and consumed during starvation, with up to 57% of the current being generated after depletion of the external carbon source. Nile blue staining of biomass samples revealed lipophilic inclusions during high substrate conditions, thus confirming the storage of polymeric material in the bacterial cells. The method also allows for indirect measurement of growth yields, which ranged from 0 to 0.54 g biomass-C formed per g substrate-C used, depending on the type of substrate and the external resistance of the circuit.

  16. Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

    PubMed Central

    Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

    2016-01-01

    Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

  17. Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression

    PubMed Central

    Lucas, Morghan C.; Timpson, Paul

    2016-01-01

    Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. PMID:27239290

  18. Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

    PubMed Central

    Özdemir, Emre; McKinney, John D.

    2015-01-01

    ABSTRACT ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. PMID:25691591

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-09-06

    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.

  3. A single-cell imaging screen reveals multiple effects of secreted small molecules on bacteria

    PubMed Central

    Salje, Jeanne

    2014-01-01

    Bacteria cells exist in close proximity to other cells of both the same and different species. Bacteria secrete a large number of different chemical species, and the local concentrations of these compounds at the surfaces of nearby cells may reach very high levels. It is fascinating to imagine how individual cells might sense and respond to the complex mix of signals at their surface. However, it is difficult to measure exactly what the local environmental composition looks like, or what the effects of individual compounds on nearby cells are. Here, an electron microscopy imaging screen was designed that would detect morphological changes induced by secreted small molecules. This differs from conventional approaches by detecting structural changes in individual cells rather than gene expression or growth rate changes at the population level. For example, one of the changes detected here was an increase in outer membrane vesicle production, which does not necessarily correspond to a change in gene expression. This initial study focussed on Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa, and revealed an intriguing range of effects of secreted small molecules on cells both within and between species. PMID:24910069

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

    PubMed Central

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

    2016-01-01

    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. PMID:27641076

  5. Epigenetic landscapes reveal transcription factors that regulate CD8(+) T cell differentiation.

    PubMed

    Yu, Bingfei; Zhang, Kai; Milner, J Justin; Toma, Clara; Chen, Runqiang; Scott-Browne, James P; Pereira, Renata M; Crotty, Shane; Chang, John T; Pipkin, Matthew E; Wang, Wei; Goldrath, Ananda W

    2017-03-13

    Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8(+) T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8(+) T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8(+) T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8(+) T cell differentiation.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27555592

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

    PubMed

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

    2015-02-06

    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.

  8. Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity

    PubMed Central

    Rulina, Anastasia V; Mittler, Frédérique; Obeid, Patricia; Gerbaud, Sophie; Guyon, Laurent; Sulpice, Eric; Kermarrec, Frédérique; Assard, Nicole; Dolega, Monika E; Gidrol, Xavier; Balakirev, Maxim Y

    2016-01-01

    Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR–ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR–ERG interaction. PMID:27906189

  9. Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

    PubMed

    Zou, Heng; Yue, Wanqing; Yu, Wai-Kin; Liu, Dandan; Fong, Chi-Chun; Zhao, Jianlong; Yang, Mengsu

    2015-07-21

    Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the β-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

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

    PubMed

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

    2017-04-01

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

  11. Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

    PubMed

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

    2016-12-20

    Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 10(6) cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 10(6) cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  12. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny

    PubMed Central

    Davis, Felicity M.; Lloyd-Lewis, Bethan; Harris, Olivia B.; Kozar, Sarah; Winton, Douglas J.; Muresan, Leila; Watson, Christine J.

    2016-01-01

    The mammary gland undergoes cycles of growth and regeneration throughout reproductive life, a process that requires mammary stem cells (MaSCs). Whilst recent genetic fate-mapping studies using lineage-specific promoters have provided valuable insights into the mammary epithelial hierarchy, the true differentiation potential of adult MaSCs remains unclear. To address this, herein we utilize a stochastic genetic-labelling strategy to indelibly mark a single cell and its progeny in situ, combined with tissue clearing and 3D imaging. Using this approach, clones arising from a single parent cell could be visualized in their entirety. We reveal that clonal progeny contribute exclusively to either luminal or basal lineages and are distributed sporadically to branching ducts or alveoli. Quantitative analyses suggest that pools of unipotent stem/progenitor cells contribute to adult mammary gland development. Our results highlight the utility of tracing a single cell and reveal that progeny of a single proliferative MaSC/progenitor are dispersed throughout the epithelium. PMID:27779190

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

    PubMed Central

    Zhang, Zheng; Christin, John R.; Wang, Chunhui; Ge, Kai; Oktay, Maja H.; Guo, Wenjun

    2016-01-01

    SUMMARY Cancer genomics have 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 GEMMs (genetically engineered mouse models). 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 established a robust in vivo platform for functional cancer genomics and discovered functional breast cancer mutations. PMID:27653681

  14. Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways

    PubMed Central

    Coleman, Ilsa; Lakely, Bryce; Coleman, Roger; Larson, Sandy; Aguirre-Ghiso, Julio A.; Xia, Jing; Gulati, Roman; Nelson, Peter S.; Montgomery, Bruce; Lange, Paul; Snyder, Linda A.; Vessella, Robert L.; Morrissey, Colm

    2014-01-01

    Cancer dormancy refers to the prolonged clinical disease-free time between removal of the primary tumor and recurrence, which is common in prostate cancer (PCa), breast cancer, esophageal cancer, and other cancers. PCa disseminated tumor cells (DTC) are detected in both patients with no evidence of disease (NED) and advanced disease (ADV). However, the molecular and cellular nature of DTC is unknown. We performed a first-in-field study of single DTC transcriptomic analyses in cancer patients to identify a molecular signature associated with cancer dormancy. We profiled eighty-five individual EpCAM+/CD45− cells from the bone marrow of PCa patients with NED or ADV. We analyzed 44 DTC with high prostate-epithelial signatures, and eliminated 41 cells with high erythroid signatures and low prostate epithelial signatures. DTC were clustered into 3 groups: NED, ADV_1, and ADV_2, in which the ADV_1 group presented a distinct gene expression pattern associated with the p38 stress activated kinase pathway. Additionally, DTC from the NED group were enriched for a tumor dormancy signature associated with head and neck squamous carcinoma and breast cancer. This study provides the first clinical evidence of the p38 pathway as a potential biomarker for early recurrence and an attractive target for therapeutic intervention. PMID:25301725

  15. Oxidant Signaling in Cells Revealed by Single Rare-Earth Based Nanoparticle Imaging

    NASA Astrophysics Data System (ADS)

    Bouzigues, Cedric; Abdesselem, Mouna; Ramodiharilafy, Rivo; Gacoin, Thierry; Tharaux, Pierre-Louis; Alexandrou, Antigoni

    The spatio-temporal organization of signaling pathways controls the cell response. Reactive oxygen species (ROS) are second messengers involved in the control of numerous normal and pathological processes and their local concentration is thus tightly regulated. However, the dynamics of ROS production and organization is mostly unknown, due to the lack of efficient probes. We developed single ROS sensitive Eu3+-doped nanoparticle imaging to quantitatively probed the intracellular ROS response. We revealed specific temporal patterns of ROS production under different types of stimulation (PDGF and ET-1) and quantitatively identified mechanisms of transactivation, which notably control the dynamics of the cell response. By using a microfluidic system, we apply spatially controlled stimulations and displayed the maintenance of asymmetric ROS concentration in the cell under a PDGF gradient. We then developed a ratiometric method using a nanoparticle mix, to quantitatively detect ROS with a 500 ms temporal resolution. We thus elucidate molecular mechanisms responsible for the control of the oxidant production kinetics. Altogether, our results reveal regulation mechanisms controlling ROS spatio-temporal organization, which can be crucial for the buildup of the cell response.

  16. Molecular analysis of aggressive renal cell carcinoma with unclassified histology reveals distinct subsets

    PubMed Central

    Chen, Ying-Bei; Xu, Jianing; Skanderup, Anders Jacobsen; Dong, Yiyu; Brannon, A. Rose; Wang, Lu; Won, Helen H.; Wang, Patricia I.; Nanjangud, Gouri J.; Jungbluth, Achim A.; Li, Wei; Ojeda, Virginia; Hakimi, A. Ari; Voss, Martin H.; Schultz, Nikolaus; Motzer, Robert J.; Russo, Paul; Cheng, Emily H.; Giancotti, Filippo G.; Lee, William; Berger, Michael F.; Tickoo, Satish K.; Reuter, Victor E.; Hsieh, James J.

    2016-01-01

    Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo–YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications. PMID:27713405

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

    PubMed Central

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

    2017-01-01

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

  18. TNFRSF13B hemizygosity reveals TACI haploinsufficiency at later stages of B-cell development

    PubMed Central

    Romberg, Neil; Virdee, Manmeet; Chamberlain, Nicolas; Oe, Tyler; Schickel, Jean-Nicolas; Perkins, Tiffany; Cantaert, Tineke; Rachid, Rima; Rosengren, Sally; Palazzo, Regina; Geha, Raif; Cunningham-Rundles, Charlotte; Meffre, Eric

    2015-01-01

    Background Heterozygous C104R or A181E TNFRSF13B mutations impair the removal of autoreactive B cells, weaken B-cell activation and convey to common variable immune deficiency (CVID) patients an increased risk for autoimmunity. How mutant TACI influences wildtype TACI function is unclear; different models suggest either a dominant-negative effect or haploinsufficiency. Objective We investigated potential TACI haploinsufficiency by analyzing antibody-deficient Smith-Magenis Syndrome (SMS) patients, who possess only one TNFRSF13B allele and antibody-deficient patients carrying one c.204insA TNFRSF13B null mutation. Methods We tested the reactivity of antibodies isolated from single B cells from SMS patients and patients with a c.204insA TNFRSF13B mutation and compared them with counterparts from CVID patients with heterozygous C104R or A181E TNFRSF13B missense mutations. We also assessed if loss of a TNFRSF13B allele induced haploinsufficiency in naïve and memory B cells recapitulate abnormal immunological features typical of CVID patients with heterozygous TNFRSF13B missense mutations. Results We found loss of a TNFRSF13B allele does not impact TACI expression, activation responses, or establishment of central B-cell tolerance in naïve B cells. Additionally, SMS patients and patients with a c.204insA TNFRSF13B mutation display normal Treg function and peripheral B-cell tolerance. The lack of a TNFRSF13B allele did result in decreased TACI expression on memory B cells, resulting in impaired activation and antibody secretion. Conclusion TNFRSF13B hemizygosity does not recapitulate autoimmune features of CVID-associated C104R and A181E TNFRSF13B mutations, which likely encode dominant-negative products, but instead reveals selective TACI haploinsufficiency at later stages of B-cell development. PMID:26100089

  19. Association of ActA to Peptidoglycan Revealed by Cell Wall Proteomics of Intracellular Listeria monocytogenes*

    PubMed Central

    García-del Portillo, Francisco; Calvo, Enrique; D'Orazio, Valentina; Pucciarelli, M. Graciela

    2011-01-01

    Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that colonizes the cytosol of eukaryotic cells. Recent transcriptomic studies have revealed that intracellular L. monocytogenes alter expression of genes encoding envelope components. However, no comparative global analysis of this cell wall remodeling process is yet known at the protein level. Here, we used high resolution mass spectrometry to define the cell wall proteome of L. monocytogenes growing inside epithelial cells. When compared with extracellular bacteria growing in a nutrient-rich medium, a major difference found in the proteome was the presence of the actin assembly-inducing protein ActA in peptidoglycan purified from intracellular bacteria. ActA was also identified in the peptidoglycan of extracellular bacteria growing in a chemically defined minimal medium. In this condition, ActA maintains its membrane anchoring domain and promotes efficient bacterial entry into nonphagocytic host cells. Unexpectedly, Internalin-A, which mediates entry of extracellular L. monocytogenes into eukaryotic cells, was identified at late infection times (6 h) as an abundant protein in the cell wall of intracellular bacteria. Other surface proteins covalently bound to the peptidoglycan, as Lmo0514 and Lmo2085, were detected exclusively in intracellular and extracellular bacteria, respectively. Altogether, these data provide the first insights into the changes occurring at the protein level in the L. monocytogenes cell wall as the pathogen transits from the extracellular environment to an intracytosolic lifestyle inside eukaryotic cells. Some of these changes include alterations in the relative amount and the mode of association of certain surface proteins. PMID:21846725

  20. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells

    NASA Technical Reports Server (NTRS)

    Hu, Shaohua; Chen, Jianxin; Fabry, Ben; Numaguchi, Yasushi; Gouldstone, Andrew; Ingber, Donald E.; Fredberg, Jeffrey J.; Butler, James P.; Wang, Ning

    2003-01-01

    We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

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

    PubMed

    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.

  2. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation

    NASA Astrophysics Data System (ADS)

    Gentleman, Eileen; Swain, Robin J.; Evans, Nicholas D.; Boonrungsiman, Suwimon; Jell, Gavin; Ball, Michael D.; Shean, Tamaryn A. V.; Oyen, Michelle L.; Porter, Alexandra; Stevens, Molly M.

    2009-09-01

    An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the `bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

  3. [Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

    PubMed

    Li, Xiao-li; Luo, Liu-bin; Hu, Xiao-qian; Lou, Bing-gan; He, Yong

    2014-06-01

    Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

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

    PubMed Central

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

    2017-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  6. ETV1 positively regulates transcription of tumor suppressor ARF

    PubMed Central

    Zynda, Evan; Jackson, Mark W; Bhattacharya, Partho; Kandel, Eugene S

    2013-01-01

    ETV1 (ETS variant 1) is a transcription factor from the ETS family and an oncogene in several types of human malignancies. Paradoxically, a predicted inactivating mutation in ETV1 was previously found in a clone of HT1080 cells with reduced activity of p53. We report that elevated expression of ETV1 makes p53-null tumor cells hypersensitive to restoration of said tumor suppressor. Furthermore, elevated levels of either wild-type ETV1 or its truncated derivative, dETV1, which mimics the product of an oncogenic rearrangement in certain tumors, results in increased expression of mRNA for p14ARF, a known activator of p53. Accordingly, expression of a luciferase reporter, which is driven by a putative ARF promoter, was elevated by concomitant expression of either ETV1 or dETV1. Our observations point to yet another example of a tumor suppressor gene being activated by a potentially oncogenic signal. A better understanding of the mechanisms that allow a cell to bypass such safeguards is needed in order to predict and prevent the development of an oncogene-tolerant state during cancer evolution. PMID:24157551

  7. Vancomycin Tolerant, Methicillin-Resistant Staphylococcus aureus Reveals the Effects of Vancomycin on Cell Wall Thickening

    PubMed Central

    Cázares-Domínguez, Vicenta; Cruz-Córdova, Ariadnna; Ochoa, Sara A.; Escalona, Gerardo; Arellano-Galindo, José; Rodríguez-Leviz, Alejandra; Hernández-Castro, Rigoberto; López-Villegas, Edgar O.; Xicohtencatl-Cortes, Juan

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/12) exhibited the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VT-MRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3% (10/12) of the isolates, and the mecIVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin. PMID:25793280

  8. Vancomycin tolerant, methicillin-resistant Staphylococcus aureus reveals the effects of vancomycin on cell wall thickening.

    PubMed

    Cázares-Domínguez, Vicenta; Cruz-Córdova, Ariadnna; Ochoa, Sara A; Escalona, Gerardo; Arellano-Galindo, José; Rodríguez-Leviz, Alejandra; Hernández-Castro, Rigoberto; López-Villegas, Edgar O; Xicohtencatl-Cortes, Juan

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/12) exhibited the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VT-MRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3% (10/12) of the isolates, and the mecIVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin.

  9. The Macronuclear Genome of Stentor coeruleus Reveals Tiny Introns in a Giant Cell.

    PubMed

    Slabodnick, Mark M; Ruby, J Graham; Reiff, Sarah B; Swart, Estienne C; Gosai, Sager; Prabakaran, Sudhakaran; Witkowska, Ewa; Larue, Graham E; Fisher, Susan; Freeman, Robert M; Gunawardena, Jeremy; Chu, William; Stover, Naomi A; Gregory, Brian D; Nowacki, Mariusz; Derisi, Joseph; Roy, Scott W; Marshall, Wallace F; Sood, Pranidhi

    2017-02-20

    The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities-if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor's cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

  10. High incidence and endemic spread of NDM-1-positive Enterobacteriaceae in Henan Province, China.

    PubMed

    Qin, Shangshang; Fu, Ying; Zhang, Qijing; Qi, Hui; Wen, Jian Guo; Xu, Hui; Xu, Lijuan; Zeng, Li; Tian, Hao; Rong, Lijuan; Li, Yonghong; Shan, Lihong; Xu, Hongde; Yu, Yunsong; Feng, Xianju; Liu, Hong-Min

    2014-08-01

    The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the bla(NDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae. Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for bla(NDM-1), and the gene was found to be carried on plasmids of various sizes (∼ 55 to ∼ 360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The bla(NDM-1)-positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each bla(NDM-1)-positive CRE isolate, the bla(NDM-1) gene was downstream of an intact ISAba125 element and upstream of the bleMBL gene. Furthermore, gene environment analysis suggested the possible transmission of bla(NDM-1)-containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca. These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread.

  11. High Incidence and Endemic Spread of NDM-1-Positive Enterobacteriaceae in Henan Province, China

    PubMed Central

    Qin, Shangshang; Fu, Ying; Zhang, Qijing; Qi, Hui; Wen, Jian Guo; Xu, Hui; Xu, Lijuan; Zeng, Li; Tian, Hao; Rong, Lijuan; Li, Yonghong; Shan, Lihong; Xu, Hongde; Yu, Yunsong

    2014-01-01

    The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the blaNDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae. Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for blaNDM-1, and the gene was found to be carried on plasmids of various sizes (∼55 to ∼360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The blaNDM-1-positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each blaNDM-1-positive CRE isolate, the blaNDM-1 gene was downstream of an intact ISAba125 element and upstream of the bleMBL gene. Furthermore, gene environment analysis suggested the possible transmission of blaNDM-1-containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca. These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread. PMID:24777095

  12. Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells.

    PubMed

    ElAzzouny, Mahmoud A; Evans, Charles R; Burant, Charles F; Kennedy, Robert T

    2015-01-01

    AMPK regulates many metabolic pathways including fatty acid and glucose metabolism, both of which are closely associated with insulin secretion in pancreatic β-cells. Insulin secretion is regulated by metabolic coupling factors such as ATP/ADP ratio and other metabolites generated by the metabolism of nutrients such as glucose, fatty acid and amino acids. However, the connection between AMPK activation and insulin secretion in β-cells has not yet been fully elucidated at a metabolic level. To study the effect of AMPK activation on glucose stimulated insulin secretion, we applied the pharmacological activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to an INS-1 (832/13) β-cell line. We measured the change in 66 metabolites in the presence or absence of AICAR using different stable isotopic labeled nutrients to probe selected pathways. AMPK activation by AICAR increased basal insulin secretion and reduced the glucose stimulation index. Although ATP/ADP ratios were not strongly affected by AICAR, several other metabolites and pathways important for insulin secretion were affected by AICAR treatment including long-chain CoAs, malonyl-CoA, 3-hydroxy-3 methylglutaryl CoA, diacylglycerol, and farnesyl pyrophosphate. Tracer studies using 13C-glucose revealed lower glucose flux in the purine and pyrimidine pathway and in the glycerolipid synthesis pathway. Untargeted metabolomics revealed reduction in ceramides caused by AICAR that may explain the beneficial role of AMPK in protecting β-cells from lipotoxicity. Taken together, the results provide an overall picture of the metabolic changes associated with AICAR treatment and how it modulates insulin secretion and β-cell survival.

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  14. Modelling epigenetic regulation of gene expression in 12 human cell types reveals combinatorial patterns of cell-type-specific genes.

    PubMed

    Lu, Yiming; Qu, Wubin; Min, Bo; Liu, Zheyan; Chen, Changsheng; Zhang, Chenggang

    2014-06-01

    The maintenance of the diverse cell types in a multicellular organism is one of the fundamental mysteries of biology. Modelling the dynamic regulatory relationships between the histone modifications and the gene expression across the diverse cell types is essential for the authors to understand the mechanisms of the epigenetic regulation. Here, the authors thoroughly assessed the histone modification enrichment profiles at the promoters and constructed quantitative models between the histone modification abundances and the gene expression in 12 human cell types. The author's results showed that the histone modifications at the promoters exhibited remarkably cell-type-dependent variability in the cell-type-specific (CTS) genes. They demonstrated that the variable profiles of the modifications are highly predictive for the dynamic changes of the gene expression across all the cell types. Their findings revealed the close relationship between the combinatorial patterns of the histone modifications and the CTS gene expression. They anticipate that the findings and the methods they used in this study could provide useful information for the future studies of the regulatory roles of the histone modifications in the CTS genes.

  15. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  16. Raman spectrum reveals the cell cycle arrest of Triptolide-induced leukemic T-lymphocytes apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Daosen; Feng, Yanyan; Zhang, Qinnan; Su, Xin; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

    2015-04-01

    Triptolide (TPL), a traditional Chinese medicine extract, possesses anti-inflammatory and anti-tumor properties. Though some research results have implicated that Triptolide (TPL) can be utilized in the treatment of leukemia, it remains controversial about the mechanism of TPL-induced leukemic T-lymphocytes apoptosis. In this study, combining Raman spectroscopic data, principal component analysis (PCA) and atomic force microscopy (AFM) imaging, both the biochemical changes and morphological changes during TPL-induced cell apoptosis were presented. In contrast, the corresponding data during Daunorubicin (DNR)-induced cell apoptosis was also exhibited. The obtained results showed that Raman spectral changes during TPL-induced cell apoptosis were greatly different from DNR-induced cell apoptosis in the early stage of apoptosis but revealed the high similarity in the late stage of apoptosis. Moreover, above Raman spectral changes were respectively consistent with the morphological changes of different stages during TPL-induced apoptosis or DNR-induced apoptosis, including membrane shrinkage and blebbing, chromatin condensation and the formation of apoptotic bodies. Importantly, it was found that Raman spectral changes with TPL-induced apoptosis or DNR-induced apoptosis were respectively related with the cell cycle G1 phase arrest or G1 and S phase arrest.

  17. Amyloid plaque structure and cell surface interactions of β-amyloid fibrils revealed by electron tomography.

    PubMed

    Han, Shen; Kollmer, Marius; Markx, Daniel; Claus, Stephanie; Walther, Paul; Fändrich, Marcus

    2017-02-27

    The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer's. This disease is characterized, if not provoked, by amyloid aggregates formed from Aβ peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aβ fibrils within a cell culture model of Alzheimer's disease. STEM images taken from the formed Aβ amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50-100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell.

  18. Systematic proteomic analysis of human hepotacellular carcinoma cells reveals molecular pathways and networks involved in metastasis.

    PubMed

    Yu, Yanyan; Shen, Huali; Yu, Hongxiu; Zhong, Fan; Zhang, Yang; Zhang, Chen; Zhao, Jian; Li, Hong; Chen, Jie; Liu, Yinkun; Yang, Pengyuan

    2011-06-01

    Systematic proteomic studying of the mechanism of hepatocellular carcinoma (HCC) metastasis remains challenging. We performed comparative proteomic and pathway analysis of four human metastatic HCC cell lines to identify metastasis-associated proteins. These HCC cell lines had a similar genetic background but with an increasing potential of metastasis. Using a combination of two dimensional electrophoresis (2-DE) and MALDI-TOF mass spectrometry, a total of 125 proteins and their post-translational modification forms or isoforms were found to be differentially expressed in the cell lines. Among them, 29 were gradually up-regulated whereas 17 were down-regulated with increasing metastatic potential. Instead of a traditional single-gene readout, global bioinformatics analysis was carried out, which revealed that the glycolysis pathway was the most significantly enriched pathway. The heat shock proteins (HSPs) centered and NF-kappaB centered networks were also enriched in the result, which may imply the key function of inflaming on metastasis. Meanwhile, knockdown of HDGF, an up-regulated protein and a target of NF-kappaB, induced cell apoptosis in the metastatic HCC cells. This work provides a demonstration that a combination of bioinformatics and comparative proteomics can help in finding out potential biomarkers associated with HCC metastasis on the level of pathways.

  19. Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish

    PubMed Central

    Gutierrez-Triana, Jose Arturo; Knerr, Boris

    2017-01-01

    Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain. PMID:28317020

  20. Amyloid plaque structure and cell surface interactions of β-amyloid fibrils revealed by electron tomography

    PubMed Central

    Han, Shen; Kollmer, Marius; Markx, Daniel; Claus, Stephanie; Walther, Paul; Fändrich, Marcus

    2017-01-01

    The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer’s. This disease is characterized, if not provoked, by amyloid aggregates formed from Aβ peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aβ fibrils within a cell culture model of Alzheimer’s disease. STEM images taken from the formed Aβ amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50–100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell. PMID:28240273

  1. Human papillomavirus infection in oral fluids of HIV-1-positive men:prevalence and risk factors

    PubMed Central

    Gaester, Karen; Fonseca, Luiz A. M.; Luiz, Olinda; Assone, Tatiane; Fontes, Adriele Souza; Costa, Fernando; Duarte, Alberto J. S.; Casseb, Jorge

    2014-01-01

    Human papillomavirus is one of the most common sexually transmitted diseases worldwide. The natural history of oral HPV infection is unclear, and its risk factors have not been explored. Immunocompromised individuals, as exemplified by HIV patients, are at high risk for HPV-related diseases. The mean of this study is to determine the prevalence ofHPV in the oral tract of HIV-1-positive male subjects and its association with risk factors. A total of 283 oral wash samples from HIV-1-positive men were tested. The oral fluid samples were used for DNA extraction and conventional PCR amplification; HPV genotyping was performed by hybridization. HPV genotyping revealed that nine samples (3.5%) were positive for HPV DNA; the major high-risk HPV types identified were 51 and 66. Worldwide studies have shown a variable prevalence of oral HPV. The diversity of genotypes and the high prevalence of multiple infections in HIV-infected subjects can be better explained by the effects of HIV-induced immunosuppression. The most important risk factors are unprotected sexual intercourse, but other factors for this infection have been described elsewhere including smoking, age and HIV-positive serostatus. In this study, smoking was the most important risk factor for acquiring oral HPV in HIV-1-infected subjects in Brazil. PMID:25322857

  2. RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival[W

    PubMed Central

    González-Ballester, David; Casero, David; Cokus, Shawn; Pellegrini, Matteo; Merchant, Sabeeha S.; Grossman, Arthur R.

    2010-01-01

    The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival. PMID:20587772

  3. Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells

    SciTech Connect

    Fromaget, Maud; Cook, Peter R. . E-mail: peter.cook@path.ox.ac.uk

    2007-08-15

    RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors - some of which are used therapeutically - affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.

  4. Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

    PubMed Central

    Hendrix, Jelle; Baumgärtel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kräusslich, Hans-Georg; Müller, Barbara

    2015-01-01

    Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly. PMID:26283800

  5. Quantitative and Temporal Requirements Revealed for Zap-70 Catalytic Activity During T Cell Development

    PubMed Central

    Au-Yeung, Byron B.; Melichar, Heather J.; Ross, Jenny O.; Cheng, Debra A.; Zikherman, Julie; Shokat, Kevan M.; Robey, Ellen A.; Weiss, Arthur

    2014-01-01

    The catalytic activity of Zap-70 is crucial for T cell receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap-70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap-70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas one hour of signaling was sufficient for negative selection. Titration of Zap-70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, revealing heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection. PMID:24908390

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

    SciTech Connect

    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.

  7. Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

    PubMed Central

    Clarke, Margaret; Maddera, Lucinda; Engel, Ulrike; Gerisch, Günther

    2010-01-01

    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/Signficance Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway. PMID:20052281

  8. Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill.

    PubMed

    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

    2012-09-01

    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.

  9. Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill

    PubMed Central

    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

    2012-01-01

    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. PMID:22717885

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

    PubMed

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

    2016-02-19

    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.

  11. Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

    PubMed

    Feldmann, Radmila; Fischer, Cornelius; Kodelja, Vitam; Behrens, Sarah; Haas, Stefan; Vingron, Martin; Timmermann, Bernd; Geikowski, Anne; Sauer, Sascha

    2013-04-01

    Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

  12. Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis

    PubMed Central

    Levine, Jacob H.; Simonds, Erin F.; Bendall, Sean C.; Davis, Kara L.; Amir, El-ad D.; Tadmor, Michelle; Litvin, Oren; Fienberg, Harris; Jager, Astraea; Zunder, Eli; Finck, Rachel; Gedman, Amanda L.; Radtke, Ina; Downing, James R.; Pe’er, Dana; Nolan, Garry P.

    2015-01-01

    SUMMARY Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology. PMID:26095251

  13. sdf1 Expression Reveals a Source of Perivascular-Derived Mesenchymal Stem Cells in Zebrafish

    PubMed Central

    Lund, Troy C.; Patrinostro, Xiaobai; Kramer, Ashley C.; Stadem, Paul; Higgins, LeeAnn; Markowski, Todd W.; Wroblewski, Matt S.; Lidke, Diane S.; Tolar, Jakub; Blazar, Bruce R.

    2014-01-01

    There is accumulating evidence that mesenchymal stem cells (MSC) have their origin as perivascular cells (PVC) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1DsRed PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by 2-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC and discovery of novel markers (CD99, CD151 and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC – MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment. PMID:24905975

  14. Sarcomere dynamics in single myocardial cells as revealed by high-resolution light diffractometry.

    PubMed

    Leung, A F

    1983-08-01

    A specially designed diffractometer with a high spatial and temporal resolution recorded the diffraction of a laser beam by single enzymatically isolated myocardial cells. The fine structures within the first-order diffraction were resolved and each structure was interpreted as the diffraction from a group of sarcomeres of nearly equal length. During activation of the cell dynamics of each discrete group of sarcomeres was uniform and independent of the other groups. However, a small nonuniform component in the sarcomere dynamics was observed and attributed to the coupling between the shortening tension and the radial stress resulting from the expansion of the myofibrillar cross-section. The time-course of the diffraction fine structures during contractile activity revealed (1) the period of the contraction-relaxation cycle, (2) the latent period, (3) the shortening and relengthening speeds and (4) the variation in the line width and intensity of the fine structure. Measurements showed that the latent period was dependent on the free Ca2+ of the cell's bathing solution while the initial shortening speed was not. The diffraction line width and intensity of the shortening cell were explained by the grating model.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation.

    PubMed

    Cicchillitti, Lucia; Manni, Isabella; Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Dell'Anna, Maria Lucia; Dell'Omo, Giulia; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Rizzo, Maria Giulia; Gurtner, Aymone; Piaggio, Giulia

    2017-01-10

    Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y-dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation.

  17. Cell Type-Specific Epigenomic Analysis Reveals a Uniquely Closed Chromatin Architecture in Mouse Rod Photoreceptors

    PubMed Central

    Hughes, Andrew E. O.; Enright, Jennifer M.; Myers, Connie A.; Shen, Susan Q.; Corbo, Joseph C.

    2017-01-01

    Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate cell types in having an ‘inverted’ nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation. PMID:28256534

  18. Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

    PubMed

    Antal, Otilia; Péter, Mária; Hackler, László; Mán, Imola; Szebeni, Gábor; Ayaydin, Ferhan; Hideghéty, Katalin; Vigh, László; Kitajka, Klára; Balogh, Gábor; Puskás, Laszló G

    2015-09-01

    Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas.

  19. Rat hepatoproliferin revealed the status of a complete hepatomitogen in human hepatoma cells.

    PubMed

    Oosthuizen, M M J; Ndaba, N; Myburgh, J A

    2005-01-01

    Hepatoproliferin (HPF), a liver regeneration factor isolated from rat hepatocytes, was assessed for its mitogenic status in the human hepatoma cell line PLC/PRF-5. HPF was able to enhance hepatoma cell growth on its own without the aid of the established complete mitogens EGF and TGF-alpha or the hepato-priming factor TNF-alpha. HPF therefore acted as a complete hepatomitogen and had no co-mitogenic properties since it did not augment proliferation when combined with EGF or TGF-alpha but showed only an additive effect in the presence of TGF-alpha. Rat HPF was phylogenetically unrestricted, because it was found active in human cells. When each of the established growth factors (GFs) was used alone, the hepatoma cells responded with the same kind of response profile, namely a bi-phasic bell-shaped dose-dependent response due to stimulation at low levels and inhibition at higher levels. However, hepatocyte growth factor (HGF) was an exception since it did not induce a growth response in hepatoma cells. On the contrary HPF, on its own, showed a progressive enhanced linear dose response at the levels used for the GFs (ie 1.0-15 ng/5 x 10(5) cells). The comparative potency (CP) (dpm x 10(3)/microg DNA/ng GF) of HPF (CP = 13) was in the same range as for the complete hepatomitogens EGF (CP = 12) and TGF-alpha (CP = 14), revealing that HPF has indeed the status of a complete mitogen.

  20. Transcription factor Fli-1 positively regulates lipopolysaccharide-induced interleukin-27 production in macrophages.

    PubMed

    Gao, Peng; Yuan, Ming; Ma, Xianwei; Jiang, Wei; Zhu, Lingxi; Wen, Mingyue; Xu, Jing; Liu, Qiuyan; An, Huazhang

    2016-03-01

    IL-27 is an important regulator of TLR4-activated innate immune. The mechanism by which IL-27 production is regulated in TLR4-activated innate immune remains largely unclear. Here we show that expression of transcription factor Fli-1 at protein level is increased in macrophages following LPS stimulation. Fli-1 overexpression increases LPS-activated IL-27 production in macrophages. Consistently, Fli-1 knockdown inhibits LPS-induced IL-27 production in macrophages. Chromatin immunoprecipitation (ChIP) assay reveals that Fli-1 binds the promoter of IL-27 p28 subunit. Further experiments manifest that Fli-1 binds the region between -250 and -150 bp upstream of the transcriptional start site of p28 gene and increases p28 gene promoter-controlled transcription. These results demonstrate that Fli-1 positively regulates IL-27 production in TLR4-activated immune response by promoting transcription of IL-27 p28 gene.

  1. A novel meta-analysis approach of cancer transcriptomes reveals prevailing transcriptional networks in cancer cells.

    PubMed

    Niida, Atsushi; Imoto, Seiya; Nagasaki, Masao; Yamaguchi, Rui; Miyano, Satoru

    2010-01-01

    Although microarray technology has revealed transcriptomic diversities underlining various cancer phenotypes, transcriptional programs controlling them have not been well elucidated. To decode transcriptional programs governing cancer transcriptomes, we have recently developed a computational method termed EEM, which searches for expression modules from prescribed gene sets defined by prior biological knowledge like TF binding motifs. In this paper, we extend our EEM approach to predict cancer transcriptional networks. Starting from functional TF binding motifs and expression modules identified by EEM, we predict cancer transcriptional networks containing regulatory TFs, associated GO terms, and interactions between TF binding motifs. To systematically analyze transcriptional programs in broad types of cancer, we applied our EEM-based network prediction method to 122 microarray datasets collected from public databases. The data sets contain about 15000 experiments for tumor samples of various tissue origins including breast, colon, lung etc. This EEM based meta-analysis successfully revealed a prevailing cancer transcriptional network which functions in a large fraction of cancer transcriptomes; they include cell-cycle and immune related sub-networks. This study demonstrates broad applicability of EEM, and opens a way to comprehensive understanding of transcriptional networks in cancer cells.

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

    PubMed Central

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

    2014-01-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure due to 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 20–30-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. PMID:25342432

  3. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells.

    PubMed

    Kowalczyk, Monika S; Tirosh, Itay; Heckl, Dirk; Rao, Tata Nageswara; Dixit, Atray; Haas, Brian J; Schneider, Rebekka K; Wagers, Amy J; Ebert, Benjamin L; Regev, Aviv

    2015-12-01

    Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs.

  4. Antiretroviral Treatment in HIV-1-Positive Mothers: Neurological Implications in Virus-Free Children

    PubMed Central

    Coelho, Antonio Victor Campos; Tricarico, Paola Maura; Celsi, Fulvio; Crovella, Sergio

    2017-01-01

    Since the worldwide introduction of antiretroviral therapy (ART) in human immunodeficiency virus type 1, HIV-1-positive mothers, together with HIV-1 testing prior to pregnancy, caesarian birth and breastfeeding cessation with replacement feeding, a reduction of HIV-1 mother-to-child transmission (MTCT) has been observed in the last few years. As such, an increasing number of children are being exposed in utero to ART. Several questions have arisen concerning the neurological effects of ART exposure in utero, considering the potential effect of antiretroviral drugs on the central nervous system, a structure which is in continuous development in the fetus and characterized by great plasticity. This review aims at discussing the possible neurological impairment of children exposed to ART in utero, focusing attention on the drugs commonly used for HIV-1 MTCT prevention, clinical reports of ART neurotoxicity in children born to HIV-1-positive mothers, and neurologic effects of protease inhibitors (PIs), especially ritonavir-“boosted” lopinavir (LPV/r) in cell and animal central nervous system models evaluating the potential neurotoxic effect of ART. Finally, we present the findings of a meta-analysis to assess the effects on the neurodevelopment of children exposed to ART in utero. PMID:28212307

  5. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    PubMed Central

    2013-01-01

    Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also

  6. Cadmium-transformed cells in the in vitro cell transformation assay reveal different proliferative behaviours and activated pathways.

    PubMed

    Forcella, M; Callegaro, G; Melchioretto, P; Gribaldo, L; Frattini, M; Stefanini, F M; Fusi, P; Urani, C

    2016-10-01

    The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1μM CdCl2 for 24h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.

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

    SciTech Connect

    Vanderslice, P.; Ballinger, S.M., Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H. )

    1990-05-01

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

  8. Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family.

    PubMed Central

    Vanderslice, P; Ballinger, S M; Tam, E K; Goldstein, S M; Craik, C S; Caughey, G H

    1990-01-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family. Images PMID:2187193

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

    PubMed

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

    2016-02-16

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

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

    PubMed

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

    2015-06-19

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

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

    PubMed Central

    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

  12. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form

    PubMed Central

    Sandoz, Kelsi M.; Popham, David L.; Beare, Paul A.; Sturdevant, Daniel E.; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A.

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3–3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3–3 cross-links as opposed to 16% 3–3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella’s environmental resistance. PMID:26909555

  13. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

    PubMed

    Sandoz, Kelsi M; Popham, David L; Beare, Paul A; Sturdevant, Daniel E; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance.

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

    PubMed

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

    2016-01-15

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

  17. Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.

    PubMed

    Bischofberger, Mirko; Iacovache, Ioan; Boss, Daniel; Naef, Felix; van der Goot, F Gisou; Molina, Nacho

    2016-04-12

    Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.

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

    PubMed Central

    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 PMID:27668431

  19. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    PubMed Central

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. Steven; Qian, Wei-Jun

    2010-01-01

    Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

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

    PubMed Central

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

    2017-01-01

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

  1. Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS

    PubMed Central

    Gopal, Angelica A.; Rappaz, Benjamin; Rouger, Vincent; Martyn, Iain B.; Dahlberg, Peter D.; Meland, Rachel J.; Beamish, Ian V.; Kennedy, Timothy E.; Wiseman, Paul W.

    2016-01-01

    Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A–D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction

  2. Morphology of primate's dopaminergic amacrine cells as revealed by TH-like immunoreactivity on retinal flat-mounts.

    PubMed

    Nguyen-Legros, J; Botteri, C; Phuc, L H; Vigny, A; Gay, M

    1984-03-12

    Dopaminergic (DA) cells have been revealed by immunohistochemical localization of tyrosine hydroxylase in the retina of cynomolgus monkey, chimpanzee and human. The DA neurons were visualized in cross-sections as well as in flat-mounts of retina. The comparison revealed a striking morphological similarity between the DA neurons in the three species. When observed in flat-mounts, they were of stellate type; when observed in cross-sections, except for a few displaced cells, they were unistratified amacrine cells branching in the outermost sublayer of the inner plexiform layer. Observations in sections suggested the existence of DA-interplexiform cells in ape and human retinas.

  3. Angiogenesis Interactome and Time Course Microarray Data Reveal the Distinct Activation Patterns in Endothelial Cells

    PubMed Central

    Chu, Liang-Hui; Lee, Esak; Bader, Joel S.; Popel, Aleksander S.

    2014-01-01

    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. PMID:25329517

  4. FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

    PubMed Central

    He, Wanzhong; Ladinsky, Mark S.; Huey-Tubman, Kathryn E.; Jensen, Grant J.; McIntosh, J. Richard; Björkman, Pamela J.

    2009-01-01

    The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers1,2, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rodents, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0-6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates efficient unidirectional transport of IgG, since FcRn binds IgG at pH 6.0-6.5 but not pH ≥7 1,2. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum, jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum)3-6. We used electron tomography to directly visualize jejunal transcytosis in space and time, developing new labeling and detection methods to map individual nanogold-labeled Fc within transport vesicles7 and to simultaneously characterize these vesicles by immunolabeling. Combining electron tomography with a non-perturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine if a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moved through networks of entangled tubular and irregular vesicles, only some of which were microtubule-associated, as it migrated to the basolateral surface. New features of transcytosis were elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis via clathrin-coated pits. Markers for early, late, and recycling endosomes each labeled vesicles in different and overlapping morphological classes, revealing unexpected spatial complexity in endo-lysosomal trafficking. PMID:18818657

  5. Emergence of NDM-1-positive capsulated Escherichia coli with high resistance to serum killing in Japan.

    PubMed

    Yamamoto, Tatsuo; Takano, Tomomi; Iwao, Yasuhisa; Hishinuma, Akira

    2011-06-01

    The New Delhi metallo-β-lactamase-1 (NDM-1) gene, bla (NDM-1), is an emerging plasmid-borne drug resistance gene, which encodes for exceptionally broad-spectrum β-lactamase, being able to hydrolyze a wide variety of β-lactams, including carbapenems, and was first reported in Klebsiella pneumoniae from a Swedish patient of Indian origin in 2009. It is widely distributed among Enterobacteriacae and has geographically exhibited extremely rapid and global spread. In this study, we characterized the bla (NDM-1)-positive ST38 Escherichia coli strain NDM-1 Dok01 (which was isolated from the blood of a 54-year-old Japanese inpatient, who had previously visited India), focusing on bacterial surface structures related to virulence. The E. coli culture contained colony variants, which developed a transparent smooth colony and a rough colony on blood agar plates. The smooth colony-forming cells (substrain M1) possessed a surface capsule and were resistant to serum killing, whereas rough colony-forming mutants (substrain B2) lacked a capsule (and a 5.3-kb plasmid) and were highly susceptible to serum killing. Reflecting the surface structural difference, substrain M1 was more flagellated and motile, whereas substrain B2 was less flagellated and apparently possessed straight pili 5 nm wide, which played a role in adherence to human intestinal cells and bacterial autoaggregation. Data suggest that the bla (NDM-1)-positive ST38 E. coli has emerged in Japan and that it is a capsulated bacterial pathogen with virulence potential in the blood stream.

  6. Dynamic changes in brewing yeast cells in culture revealed by statistical analyses of yeast morphological data.

    PubMed

    Ohnuki, Shinsuke; Enomoto, Kenichi; Yoshimoto, Hiroyuki; Ohya, Yoshikazu

    2014-03-01

    The vitality of brewing yeasts has been used to monitor their physiological state during fermentation. To investigate the fermentation process, we used the image processing software, CalMorph, which generates morphological data on yeast mother cells and bud shape, nuclear shape and location, and actin distribution. We found that 248 parameters changed significantly during fermentation. Successive use of principal component analysis (PCA) revealed several important features of yeast, providing insight into the dynamic changes in the yeast population. First, PCA indicated that much of the observed variability in the experiment was summarized in just two components: a change with a peak and a change over time. Second, PCA indicated the independent and important morphological features responsible for dynamic changes: budding ratio, nucleus position, neck position, and actin organization. Thus, the large amount of data provided by imaging analysis can be used to monitor the fermentation processes involved in beer and bioethanol production.

  7. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours.

    PubMed

    Litchfield, Kevin; Summersgill, Brenda; Yost, Shawn; Sultana, Razvan; Labreche, Karim; Dudakia, Darshna; Renwick, Anthony; Seal, Sheila; Al-Saadi, Reem; Broderick, Peter; Turner, Nicholas C; Houlston, Richard S; Huddart, Robert; Shipley, Janet; Turnbull, Clare

    2015-01-22

    Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT.

  8. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours

    PubMed Central

    Litchfield, Kevin; Summersgill, Brenda; Yost, Shawn; Sultana, Razvan; Labreche, Karim; Dudakia, Darshna; Renwick, Anthony; Seal, Sheila; Al-Saadi, Reem; Broderick, Peter; Turner, Nicholas C.; Houlston, Richard S.; Huddart, Robert; Shipley, Janet; Turnbull, Clare

    2015-01-01

    Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT. PMID:25609015

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA

    PubMed Central

    Gremel, G.; Lee, R. J.; Girotti, M. R.; Mandal, A. K.; Valpione, S.; Garner, G.; Ayub, M.; Wood, S.; Rothwell, D. G.; Fusi, A.; Wallace, A.; Brady, G.; Dive, C.; Dhomen, N.; Lorigan, P.; Marais, R.

    2016-01-01

    Background The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. Patients and methods We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. Results Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. Conclusion We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available. PMID:27502704

  11. Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential

    PubMed Central

    Bolton, Helen; Graham, Sarah J. L.; Van der Aa, Niels; Kumar, Parveen; Theunis, Koen; Fernandez Gallardo, Elia; Voet, Thierry; Zernicka-Goetz, Magdalena

    2016-01-01

    Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic. PMID:27021558

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

    PubMed

    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.

  13. High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

    PubMed Central

    Schmid, Benjamin; Shah, Gopi; Scherf, Nico; Weber, Michael; Thierbach, Konstantin; Campos, Citlali Pérez; Roeder, Ingo; Aanstad, Pia; Huisken, Jan

    2013-01-01

    The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology. PMID:23884240

  14. Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery

    PubMed Central

    Jarvela, Timothy; Linstedt, Adam D.

    2012-01-01

    Acute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general gene-based strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Light-induced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex. PMID:22421362

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27461848

  17. Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

    PubMed Central

    Oikawa, Tsunekazu; Wauthier, Eliane; Dinh, Timothy A.; Selitsky, Sara R.; Reyna-Neyra, Andrea; Carpino, Guido; Levine, Ronald; Cardinale, Vincenzo; Klimstra, David; Gaudio, Eugenio; Alvaro, Domenico; Carrasco, Nancy; Sethupathy, Praveen; Reid, Lola M.

    2015-01-01

    The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. PMID:26437858

  18. Correction: Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    PubMed Central

    2014-01-01

    . Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also revealed a complex history of lineage-specific expansions and attritions for the PL1 family. Conclusions Our study provides insights into the variety and expansion of fungal CAZyme classes and revealed the relationship of CAZyme size and diversity with their nutritional strategy and host specificity. PMID:24422981

  19. Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2.

    PubMed

    Mussotter, Franz; Tomm, Janina Melanie; El Ali, Zeina; Pallardy, Marc; Kerdine-Römer, Saadia; Götz, Mario; von Bergen, Martin; Haase, Andrea; Luch, Andreas

    2016-12-15

    Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2(+/+)) and Nrf2 knockout (nrf2(-/-)) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100μM CA, respectively. For 5 and 10μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2(-/-) BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization.

  20. Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

    PubMed Central

    2013-01-01

    Background The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet. Results We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules. Conclusions We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host. PMID:24286338

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

    PubMed

    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.

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

    PubMed

    Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Peng, Shih-Jung; Tang, Shiue-Cheng

    2015-02-01

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

  3. Value of Perfusion-Weighted MR Imaging in the Assessment of Early Cerebral Alterations in Neurologically Asymptomatic HIV-1-Positive and HCV-Positive Patients

    PubMed Central

    Bladowska, Joanna; Knysz, Brygida; Zimny, Anna; Małyszczak, Krzysztof; Kołtowska, Anna; Szewczyk, Paweł; Gąsiorowski, Jacek; Furdal, Michał; Sąsiadek, Marek J.

    2014-01-01

    BG in HCV-positive patients (p = 0.0002; p<0.0001) compared to controls as well as to all HIV-1-positive subjects. There were no significant correlations of rCBV values and CD4 T cell count or HAI score. Conclusions PWI examination enables the assessment of HIV-related as well as HCV-related early cerebral dysfunction in asymptomatic subjects. HCV-infected patients seem to reveal the most pronounced perfusion changes. PMID:25013963

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

    PubMed

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

    2016-02-04

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    Resch, M.

    2014-09-08

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

  7. Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair

    PubMed Central

    Pipalia, Tapan G.; Koth, Jana; Roy, Shukolpa D.; Hammond, Christina L.; Kawakami, Koichi

    2016-01-01

    ABSTRACT Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7+ cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular

  8. Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

    PubMed Central

    György, Bence; Szabó, Tamás G.; Turiák, Lilla; Wright, Matthew; Herczeg, Petra; Lédeczi, Zsigmond; Kittel, Ágnes; Polgár, Anna; Tóth, Kálmán; Dérfalvi, Beáta; Zelenák, Gergő; Böröcz, István; Carr, Bob; Nagy, György; Vékey, Károly; Gay, Steffen; Falus, András; Buzás, Edit I.

    2012-01-01

    Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. PMID:23185418

  9. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions.

    PubMed

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N; Flood, Beverly E; Bailey, Jake V; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  10. Exon expression profiling reveals stimulus-mediated exon use in neural cells

    PubMed Central

    McKee, Adrienne E; Neretti, Nicola; Carvalho, Luis E; Meyer, Clifford A; Fox, Edward A; Brodsky, Alexander S; Silver, Pamela A

    2007-01-01

    Background: Neuronal cells respond to changes in intracellular calcium ([Ca2+]i) by affecting both the abundance and architecture of specific mRNAs. Although calcium-induced transcription and transcript variation have both been recognized as important sources of gene regulation, the interplay between these two phenomena has not been evaluated on a genome-wide scale. Results: Here, we show that exon-centric microarrays can be used to resolve the [Ca2+]i-modulated gene expression response into transcript-level and exon-level regulation. Global assessments of affected transcripts reveal modulation within distinct functional gene categories. We find that transcripts containing calcium-modulated exons exhibit enrichment for calcium ion binding, calmodulin binding, plasma membrane associated, and metabolic proteins. Additionally, we uncover instances of regulated exon use in potassium channels, neuroendocrine secretory proteins and metabolic enzymes, and demonstrate that regulated changes in exon expression give rise to distinct transcript variants. Conclusion: Our findings connect extracellular stimuli to specific exon behavior, and suggest that changes in transcript and exon abundance are reflective of a coordinated gene expression response to elevated [Ca2+]i. The technology we describe here lends itself readily to the resolution of stimulus-induced gene expression at both the transcript and exon levels. PMID:17683528

  11. Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma.

    PubMed

    Wu, Chih-Ching; Chu, Hao-Wei; Hsu, Chia-Wei; Chang, Kai-Ping; Liu, Hao-Ping

    2015-10-01

    Oral cavity squamous cell carcinoma (OSCC), which is frequently associated with poor prognosis and mortality, is a leading cause of cancer-related death worldwide. Discovery of body fluid accessible biomarkers is needed to improve OSCC screening. To this end, we profiled proteomes of saliva from the healthy volunteers, the individuals with oral potentially malignant disorders (OPMD), and the OSCC patients by means of SDS-PAGE coupled with LC-MS/MS. In the control, the OPMD, and the OSCC groups, 958, 845, and 1030 salivary proteins were detected, respectively. With spectral counting-based label-free quantification, 22 overexpressed salivary proteins were identified in the OSCC group compared with the healthy controls and the OPMD individuals. Among them, resistin (RETN) was subjected to further validation with an independent cohort using ELISA. The data confirmed that the salivary RETN levels in the OSCC patients were significantly higher than that in the healthy or in the OPMD group. Moreover, the elevated levels of salivary RETN were highly correlated with late-stage primary tumors, advanced overall stage, and lymph-node metastasis. Our results not only reveal that profiling of saliva proteome is feasible for discovery of OSCC biomarkers, but also identify RETN as a potential salivary biomarker for OSCC detection.

  12. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

    PubMed Central

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

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

    SciTech Connect

    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.

  14. A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

    PubMed

    Torres-Puig, Sergi; Broto, Alicia; Querol, Enrique; Piñol, Jaume; Pich, Oscar Q

    2015-05-26

    The Mycoplasma genitalium MG428 protein shows homology to members of the sigma-70 family of sigma factors. Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function. Deletion of MG_428 or some of the up-regulated unknown genes led to severe recombination defects. Single cell analyses revealed that activation of the MG428-regulon is a rare event under laboratory growth conditions. A conserved sequence with sigma-70 promoter architecture (TTGTCA-N(18/19)-ATTWAT) was identified in the upstream region of all of the MG428-regulated genes or operons. Primer extension analyses demonstrated that transcription initiates immediately downstream of this sigma70-type promoter in a MG428-dependent manner. Furthermore, mutagenesis of the conserved -10 and -35 elements corroborated the requirement of these regions for promoter function. Therefore, a new mycoplasma promoter directs transcription of a unique recombination regulon. Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor. Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism. Since recombination is an important mechanism to generate antigenic variation, MG428 emerges as a novel factor contributing to M. genitalium virulence.

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

    PubMed Central

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

    2014-01-01

    The somatosensory nervous system is critical for the organism's ability to respond to mechanical, thermal, and nociceptive stimuli. Somatosensory neurons are functionally and anatomically diverse but their molecular profiles are not well-defined. Here, we used transcriptional profiling to analyze the detailed molecular signatures of dorsal root ganglion (DRG) sensory neurons. We used two mouse reporter lines and surface IB4 labeling to purify three major non-overlapping classes of neurons: 1) IB4+SNS-Cre/TdTomato+, 2) IB4−SNS-Cre/TdTomato+, and 3) Parv-Cre/TdTomato+ cells, encompassing the majority of nociceptive, pruriceptive, and proprioceptive neurons. These neurons displayed distinct expression patterns of ion channels, transcription factors, and GPCRs. Highly parallel qRT-PCR analysis of 334 single neurons selected by membership of the three populations demonstrated further diversity, with unbiased clustering analysis identifying six distinct subgroups. These data significantly increase our knowledge of the molecular identities of known DRG populations and uncover potentially novel subsets, revealing the complexity and diversity of those neurons underlying somatosensation. DOI: http://dx.doi.org/10.7554/eLife.04660.001 PMID:25525749

  16. Long-term artificial selection reveals a role of TCTP in autophagy in mammalian cells.

    PubMed

    Chen, Ke; Huang, Chunhua; Yuan, Jia; Cheng, Hanhua; Zhou, Rongjia

    2014-08-01

    Understanding genomic variation and detecting selection signatures in a genome under selection have been great challenges for a century. Activation, development/exhaustion of primordial follicles in mammalian ovary determines reproductive success, menopause/end of female reproductive life. However, molecular mechanisms underlying oogenesis, particularly under artificial selection, are largely unknown. We report that a proteome-wide scan for selection signatures in the genome over 9,000 years of artificial pressure on the ovary revealed a general picture of selection signatures in the genome, especially genomic variations through artificial selection were detected in promoter and intron regions. Crossbreeding between domestic and wild species results in more than half of the protein spots exhibiting heterosis. Translationally controlled tumor protein (TCTP) is upregulated by artificial selection and positively regulates autophagy through the AMP-activated protein kinase pathway. Notably, TCTP interacts with ATG16 complex. In addition to cytoplasmic autophagy, nucleophagy occurs in the nuclei of granulosa and cumulus cells in ovaries, indicating an importance of the nuclear material for degradation by nucleophagy. Our findings provide insight into cellular and molecular mechanisms relevant for improvement of ovary functions, and identify selection signatures in the genome for ovary function over long-term artificial selection pressure.

  17. Expression profiling of constitutive mast cells reveals a unique identity within the immune system

    PubMed Central

    Dwyer, Daniel F.; Barrett, Nora A.; Austen, K. Frank

    2016-01-01

    Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity IgE receptor and are implicated in host defense and diverse immune-mediated diseases. To better characterize the function of these cells, we assessed the transcriptional profiles of mast cells isolated from peripheral connective tissues and basophils isolated from spleen and blood. We found that mast cells were transcriptionally distinct, clustering independently from all other profiled cells, and that mast cells demonstrated considerably greater heterogeneity across tissues than previously appreciated. We observed minimal homology between mast cells and basophils, which share more overlap with other circulating granulocytes than with mast cells. Derivation of mast cell and basophil transcriptional signatures underscores their differential capacity to detect environmental signals and influence the inflammatory milieu. PMID:27135604

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

    PubMed

    Ritsma, Laila; Ellenbroek, Saskia I J; Zomer, Anoek; Snippert, Hugo J; de Sauvage, Frederic J; Simons, Benjamin D; Clevers, Hans; van Rheenen, Jacco

    2014-03-20

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

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

    NASA Astrophysics Data System (ADS)

    Ritsma, Laila; Ellenbroek, Saskia I. J.; Zomer, Anoek; Snippert, Hugo J.; de Sauvage, Frederic J.; Simons, Benjamin D.; Clevers, Hans; van Rheenen, Jacco

    2014-03-01

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

  20. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells

    PubMed Central

    Armitage, Emily G.; Kotze, Helen L.; Allwood, J. William; Dunn, Warwick B.; Goodacre, Royston; Williams, Kaye J.

    2015-01-01

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments. PMID:26508589

  1. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells.

    PubMed

    Armitage, Emily G; Kotze, Helen L; Allwood, J William; Dunn, Warwick B; Goodacre, Royston; Williams, Kaye J

    2015-10-28

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments.

  2. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    PubMed

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  3. Single-cell sequencing of Thiomargarita reveals genomic flexibility for adaptation to dynamic redox conditions

    DOE PAGES

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; ...

    2016-06-21

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiornargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of amore » chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In conclusion, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur

  4. Single-Cell (Meta-)Genomics of a Dimorphic Candidatus Thiomargarita nelsonii Reveals Genomic Plasticity

    PubMed Central

    Flood, Beverly E.; Fliss, Palmer; Jones, Daniel S.; Dick, Gregory J.; Jain, Sunit; Kaster, Anne-Kristin; Winkel, Matthias; Mußmann, Marc; Bailey, Jake

    2016-01-01

    The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group

  5. Single-cell Sequencing of Thiomargarita Reveals Genomic Flexibility for Adaptation to Dynamic Redox Conditions

    PubMed Central

    Winkel, Matthias; Salman-Carvalho, Verena; Woyke, Tanja; Richter, Michael; Schulz-Vogt, Heide N.; Flood, Beverly E.; Bailey, Jake V.; Mußmann, Marc

    2016-01-01

    Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming “Candidatus Thiomargarita nelsonii Thio36”, and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. “Ca. T. nelsonii Thio36” is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that “Ca. T. nelsonii Thio36” can function as a chemolithoautotroph. Carbon can be fixed via the Calvin–Benson–Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, “Ca. T. nelsonii Thio36” also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na+-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae “Ca. T. nelsonii Thio36” encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of “Ca. T. nelsonii Thio36” provides additional insight into the ecology of

  6. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.

    PubMed

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-11-01

    Blood-brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0 · 05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings.

  7. Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells

    PubMed Central

    Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

    2014-01-01

    Blood–brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0·05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. PMID:24801999

  8. 4D Traction Force Microscopy Reveals Asymmetric Cortical Forces in Migrating Dictyostelium Cells

    NASA Astrophysics Data System (ADS)

    Delanoë-Ayari, H.; Rieu, J. P.; Sano, M.

    2010-12-01

    We present a 4D (x; y; z; t) force map of Dictyostelium cells crawling on a soft gel substrate. Vertical forces are of the same order as the tangential ones. The cells pull the substratum upward along the cell, medium, or substratum contact line and push it downward under the cell except for the pseudopods. We demonstrate quantitatively that the variations in the asymmetry in cortical forces correlates with the variations of the direction and speed of cell displacement.

  9. The CWB2 Cell Wall-Anchoring Module Is Revealed by the Crystal Structures of the Clostridium difficile Cell Wall Proteins Cwp8 and Cwp6.

    PubMed

    Usenik, Aleksandra; Renko, Miha; Mihelič, Marko; Lindič, Nataša; Borišek, Jure; Perdih, Andrej; Pretnar, Gregor; Müller, Uwe; Turk, Dušan

    2017-03-07

    Bacterial cell wall proteins play crucial roles in cell survival, growth, and environmental interactions. In Gram-positive bacteria, cell wall proteins include several types that are non-covalently attached via cell wall binding domains. Of the two conserved surface-layer (S-layer)-anchoring modules composed of three tandem SLH or CWB2 domains, the latter have so far eluded structural insight. The crystal structures of Cwp8 and Cwp6 reveal multi-domain proteins, each containing an embedded CWB2 module. It consists of a triangular trimer of Rossmann-fold CWB2 domains, a feature common to 29 cell wall proteins in Clostridium difficile 630. The structural basis of the intact module fold necessary for its binding to the cell wall is revealed. A comparison with previously reported atomic force microscopy data of S-layers suggests that C. difficile S-layers are complex oligomeric structures, likely composed of several different proteins.

  10. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells

    PubMed Central

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-01

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas. PMID:28059107

  11. A comparative mechanical analysis of plant and animal cells reveals convergence across kingdoms.

    PubMed

    Durand-Smet, Pauline; Chastrette, Nicolas; Guiroy, Axel; Richert, Alain; Berne-Dedieu, Annick; Szecsi, Judit; Boudaoud, Arezki; Frachisse, Jean-Marie; Bendahmane, Mohammed; Bendhamane, Mohammed; Hamant, Oliver; Asnacios, Atef

    2014-11-18

    Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.

  12. M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens.

    PubMed

    Coscolla, Mireia; Copin, Richard; Sutherland, Jayne; Gehre, Florian; de Jong, Bouke; Owolabi, Olumuiya; Mbayo, Georgetta; Giardina, Federica; Ernst, Joel D; Gagneux, Sebastien

    2015-11-11

    Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.

  13. Gene expression kinetics in individual plasmodial cells reveal alternative programs of differential regulation during commitment and differentiation.

    PubMed

    Rätzel, Viktoria; Marwan, Wolfgang

    2015-05-26

    During its life cycle, the amoebozoon Physarum polycephalum forms multinucleate plasmodial cells that can grow to macroscopic size while maintaining a naturally synchronous population of nuclei. Sporulation-competent plasmodia were stimulated through photoactivation of the phytochrome photoreceptor and the expression of sporulation marker genes was analyzed quantitatively by repeatedly taking samples of the same plasmodial cell at successive time points after the stimulus pulse. Principal component analysis of the gene expression data revealed that plasmodial cells take different trajectories leading to cell fate decision and differentiation and suggested that averaging over individual cells is inappropriate. Queries for genes with pairwise correlated expression kinetics revealed qualitatively different patterns of co-regulation, indicating that alternative programs of differential regulation are operational in individual plasmodial cells. At the single cell level, the response to stimulation of a non-sporulating mutant was qualitatively different as compared to the wild type with respect to the differentially regulated genes and their patterns of co-regulation. The observation of individual differences during commitment and differentiation supports the concept of a Waddington-type quasipotential landscape for the regulatory control of cell differentiation. Comparison of wild type and sporulation mutant data further supports the idea that mutations may impact the topology of this landscape.

  14. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction.

    PubMed

    Lodie, Tracey A; Blickarz, Courtney E; Devarakonda, Tara J; He, Chufa; Dash, Ajeeta B; Clarke, Jennifer; Gleneck, Kristen; Shihabuddin, Lamya; Tubo, Ross

    2002-10-01

    Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

  15. Complete sequences of two plasmids in a blaNDM-1-positive Klebsiella oxytoca isolate from Taiwan.

    PubMed

    Huang, Tzu-Wen; Wang, Jann-Tay; Lauderdale, Tsai-Ling; Liao, Tsai-Lien; Lai, Jui-Fen; Tan, Mei-Chen; Lin, Ann-Chi; Chen, Ying-Tsong; Tsai, Shih-Feng; Chang, Shan-Chwen

    2013-08-01

    Genetic determinants of a bla(NDM-1)-positive, multidrug-resistant bacterial isolate that caused active infection was investigated by DNA sequencing. Two plasmids, pKOX_NDM1 and pKOX-R1, were identified for the Klebsiella oxytoca strain E718. Sequence annotation revealed a bla(NDM-1) gene in pKOX_NDM1 and two extended-spectrum β-lactamase producers (bla(CTX-M-3) and blaSHV-12) and a wide array of resistance genes in pKOX-R1. These findings highlight the difficulty in treating multidrug-resistant bacterial infections and the potential danger of emerging resistant enterobacteria.

  16. Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

    PubMed Central

    Wilkins, Christopher E.; Marlow, Laura A.; Malyarenko, Dariya; Kim, Yunee; Ignatchenko, Alexandr; Sasinowska, Heather; Sasinowski, Maciek; Nyalwidhe, Julius O.; Kislinger, Thomas; Copland, John A.; Drake, Richard R.

    2016-01-01

    Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from

  17. RNA-Seq Reveals the Angiogenesis Diversity between the Fetal and Adults Bone Mesenchyme Stem Cell.

    PubMed

    Zhao, Xin; Han, Yingmin; Liang, Yu; Nie, Chao; Wang, Jian

    2016-01-01

    In this research, we used RNA sequencing (RNA-seq) to analyze 23 single cell samples and 2 bulk cells sample from human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. The results from the research demonstrated that there were big differences between two cell lines. Adult bone mesenchyme stem cell lines showed a strong trend on the blood vessel differentiation and cell motion, 48/49 vascular related differential expressed genes showed higher expression in adult bone mesenchyme stem cell lines (Abmsc) than fetal bone mesenchyme stem cell lines (Fbmsc). 96/106 cell motion related genes showed the same tendency. Further analysis showed that genes like ANGPT1, VEGFA, FGF2, PDGFB and PDGFRA showed higher expression in Abmsc. This work showed cell heterogeneity between human adult bone mesenchyme stem cell line and human fetal bone mesenchyme stem cell line. Also the work may give an indication that Abmsc had a better potency than Fbmsc in the future vascular related application.

  18. Cross-contamination of cell lines as revealed by DNA fingerprinting in the IFO animal cell bank.

    PubMed

    Satoh, M; Takeuchi, M

    1993-01-01

    For quality control of cell lines, the Institute for Fermentation, Osaka (IFO) animal cell bank recently introduced DNA fingerprinting analysis, which enables verification of cell lines at the individual level, to detect cross-culture contamination. By using this analysis, we found two cases of cross-contamination of cell lines.

  19. Single cell transcriptomics reveals specific RNA editing signatures in the human brain.

    PubMed

    Picardi, Ernesto; Horner, David Stephen; Pesole, Graziano

    2017-03-03

    While RNA editing by A-to-I deamination is a requisite for neuronal function in humans, it is under investigated in single cells. Here we fill this gap by analysing RNA editing profiles of single cells from the brain cortex of living human subjects. We show that RNA editing levels per cell are bimodally distributed and distinguish between major brain cell types thus providing new insights into neuronal dynamics.

  20. Dynamic physical properties of dissociated tumor cells revealed by dielectrophoretic field-flow fractionation

    PubMed Central

    Shim, Sangjo; Gascoyne, Peter; Noshari, Jamileh; Stemke Hale, Katherine

    2013-01-01

    Metastatic disease results from the shedding of cancer cells from a solid primary tumor, their transport through the cardiovascular system as circulating tumor cells (CTCs) and their engraftment and growth at distant sites. Little is known about the properties and fate of tumor cells as they leave their growth site and travel as single cells. We applied analytical dielectrophoretic field-flow fractionation (dFFF) to study the membrane capacitance, density and hydrodynamic properties together with the size and morphology of cultured tumor cells after they were harvested and placed into single cell suspensions. After detachment, the tumor cells exhibited biophysical properties that changed with time through a process of cytoplasmic shedding whereby membrane and cytoplasm were lost. This process appeared to be distinct from the cell death mechanisms of apoptosis, anoikis and necrosis and it may explain why multiple phenotypes are seen among CTCs isolated from patients and among the tumor cells obtained from ascitic fluid of patients. The implications of dynamic biophysical properties and cytoplasmic loss for CTC migration into small blood vessels in the circulatory system, survival and gene expression are discussed. Because the total capacitance of tumor cells remained higher than blood cells even after they had shed cytoplasm, dFFF offers a compelling, antibody-independent technology for isolating viable CTCs from blood even when they are no larger than peripheral blood mononuclear cells. PMID:21691666

  1. Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics

    PubMed Central

    D'Aurizio, Federica; Puppato, Elisa; Pandolfi, Maura; Beltrami, Antonio Paolo; Cesselli, Daniela; Falini, Giuseppe; Beltrami, Carlo Alberto; Curcio, Francesco

    2012-01-01

    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. PMID:22844522

  2. Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

    PubMed Central

    Choong, Meng Ling; Tan, Shan Ho; Tan, Tuan Zea; Manesh, Sravanthy; Ngo, Anna; Yong, Jacklyn W. Y.; Yang, Henry He; Lee, May Ann

    2014-01-01

    Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. PMID:25343454

  3. A Comparative Transcriptomic Analysis Reveals Conserved Features of Stem Cell Pluripotency in Planarians and Mammals

    PubMed Central

    Labbé, Roselyne M.; Irimia, Manuel; Currie, Ko W.; Lin, Alexander; Zhu, Shu Jun; Brown, David D.R.; Ross, Eric J.; Voisin, Veronique; Bader, Gary D.; Blencowe, Benjamin J.; Pearson, Bret J.

    2014-01-01

    Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency. PMID:22696458

  4. A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals.

    PubMed

    Labbé, Roselyne M; Irimia, Manuel; Currie, Ko W; Lin, Alexander; Zhu, Shu Jun; Brown, David D R; Ross, Eric J; Voisin, Veronique; Bader, Gary D; Blencowe, Benjamin J; Pearson, Bret J

    2012-08-01

    Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency.

  5. Novel middle-type Kenyon cells in the honeybee brain revealed by area-preferential gene expression analysis.

    PubMed

    Kaneko, Kumi; Ikeda, Tsubomi; Nagai, Mirai; Hori, Sayaka; Umatani, Chie; Tadano, Hiroto; Ugajin, Atsushi; Nakaoka, Takayoshi; Paul, Rajib Kumar; Fujiyuki, Tomoko; Shirai, Kenichi; Kunieda, Takekazu; Takeuchi, Hideaki; Kubo, Takeo

    2013-01-01

    The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel 'middle-type Kenyon cells' that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large- and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large- and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large- and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a 'clustering' of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.

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

    NASA Astrophysics Data System (ADS)

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

    1992-09-01

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

  7. TGF-β stimulation in human and murine cells reveals commonly affected biological processes and pathways at transcription level

    PubMed Central

    2014-01-01

    Background The TGF-β signaling pathway is a fundamental pathway in the living cell, which plays a key role in many central cellular processes. The complex and sometimes contradicting mechanisms by which TGF-β yields phenotypic effects are not yet completely understood. In this study we investigated and compared the transcriptional response profile of TGF-β1 stimulation in different cell types. For this purpose, extensive experiments are performed and time-course microarray data are generated in human and mouse parenchymal liver cells, human mesenchymal stromal cells and mouse hematopoietic progenitor cells at different time points. We applied a panel of bioinformatics methods on our data to uncover common patterns in the dynamic gene expression response in respective cells. Results Our analysis revealed a quite variable and multifaceted transcriptional response profile of TGF-β1 stimulation, which goes far beyond the well-characterized classical TGF-β1 signaling pathway. Nonetheless, we could identify several commonly affected processes and signaling pathways across cell types and species. In addition our analysis suggested an important role of the transcription factor EGR1, which appeared to have a conserved influence across cell-types and species. Validation via an independent dataset on A549 lung adenocarcinoma cells largely confirmed our findings. Network analysis suggested explanations, how TGF-β1 stimulation could lead to the observed effects. Conclusions The analysis of dynamical transcriptional response to TGF-β treatment experiments in different human and murine cell systems revealed commonly affected biological processes and pathways, which could be linked to TGF-β1 via network analysis. This helps to gain insights about TGF-β pathway activities in these cell systems and its conserved interactions between the species and tissue types. PMID:24886091

  8. Direct laser manipulation reveals the mechanics of cell contacts in vivo

    PubMed Central

    Bambardekar, Kapil; Clément, Raphaël; Blanc, Olivier; Chardès, Claire; Lenne, Pierre-François

    2015-01-01

    Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue. PMID:25605934

  9. Functional Sphere Profiling Reveals the Complexity of Neuroblastoma Tumor-Initiating Cell Model12

    PubMed Central

    Coulon, Aurélie; Flahaut, Marjorie; Mühlethaler-Mottet, Annick; Meier, Roland; Liberman, Julie; Balmas-Bourloud, Katia; Nardou, Katya; Yan, Pu; Tercier, Stéphane; Joseph, Jean-Marc; Sommer, Lukas; Gross, Nicole

    2011-01-01

    Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically “profile” the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB. PMID:22028624

  10. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    PubMed Central

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Zhang, Qing; Choi, Sung W.; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA–transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  11. Computational modelling of cell chain migration reveals mechanisms that sustain follow-the-leader behaviour.

    PubMed

    Wynn, Michelle L; Kulesa, Paul M; Schnell, Santiago

    2012-07-07

    Follow-the-leader chain migration is a striking cell migratory behaviour observed during vertebrate development, adult neurogenesis and cancer metastasis. Although cell-cell contact and extracellular matrix (ECM) cues have been proposed to promote this phenomenon, mechanisms that underlie chain migration persistence remain unclear. Here, we developed a quantitative agent-based modelling framework to test mechanistic hypotheses of chain migration persistence. We defined chain migration and its persistence based on evidence from the highly migratory neural crest model system, where cells within a chain extend and retract filopodia in short-lived cell contacts and move together as a collective. In our agent-based simulations, we began with a set of agents arranged as a chain and systematically probed the influence of model parameters to identify factors critical to the maintenance of the chain migration pattern. We discovered that chain migration persistence requires a high degree of directional bias in both lead and follower cells towards the target. Chain migration persistence was also promoted when lead cells maintained cell contact with followers, but not vice-versa. Finally, providing a path of least resistance in the ECM was not sufficient alone to drive chain persistence. Our results indicate that chain migration persistence depends on the interplay of directional cell movement and biased cell-cell contact.

  12. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    SciTech Connect

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  13. Systematic drug perturbations on cancer cells reveal diverse exit paths from proliferative state

    PubMed Central

    Xiao, Caide; Rubin, Irit; Kauffman, Stuart A.; Schroeder, Michael; Huang, Sui

    2016-01-01

    During a cell state transition, cells travel along trajectories in a gene expression state space. This dynamical systems framework complements the traditional concept of molecular pathways that drive cell phenotype switching. To expose the structure that hinders cancer cells from exiting robust proliferative state, we assessed the perturbation capacity of a drug library and identified 16 non-cytotoxic compounds that stimulate MCF7 breast cancer cells to exit from proliferative state to differentiated state. The transcriptome trajectories triggered by these drugs diverged, then converged. Chemical structures and drug targets of these compounds overlapped minimally. However, a network analysis of targeted pathways identified a core signaling pathway - indicating common stress-response and down-regulation of STAT1 before differentiation. This multi-trajectory analysis explores the cells' state transition with a multitude of perturbations in combination with traditional pathway analysis, leading to an encompassing picture of the dynamics of a therapeutically desired cell-state switching. PMID:26871731

  14. Genome sequencing of normal cells reveals developmental lineages and mutational processes

    PubMed Central

    Behjati, Sam; Wedge, David C; Tamuri, Asif U; Martincorena, Inigo; Petljak, Mia; Alexandrov, Ludmil B; Gundem, Gunes; Tarpey, Patrick S; Roerink, Sophie; Blokker, Joyce; Maddison, Mark; Mudie, Laura; Robinson, Ben; Nik-Zainal, Serena; Campbell, Peter; Goldman, Nick; van de Wetering, Marc; Cuppen, Edwin; Clevers, Hans; Stratton, Michael R

    2014-01-01

    The somatic mutations present in the genome of a cell have been accumulated over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree1, the number of divisions each cell has undergone and the mutational processes that have been operative2. Here, we conducted whole genome sequencing of clonal lines3 derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible. PMID:25043003

  15. Systematic drug perturbations on cancer cells reveal diverse exit paths from proliferative state.

    PubMed

    Zhou, Joseph X; Isik, Zerrin; Xiao, Caide; Rubin, Irit; Kauffman, Stuart A; Schroeder, Michael; Huang, Sui

    2016-02-16

    During a cell state transition, cells travel along trajectories in a gene expression state space. This dynamical systems framework complements the traditional concept of molecular pathways that drive cell phenotype switching. To expose the structure that hinders cancer cells from exiting robust proliferative state, we assessed the perturbation capacity of a drug library and identified 16 non-cytotoxic compounds that stimulate MCF7 breast cancer cells to exit from proliferative state to differentiated state. The transcriptome trajectories triggered by these drugs diverged, then converged. Chemical structures and drug targets of these compounds overlapped minimally. However, a network analysis of targeted pathways identified a core signaling pathway--indicating common stress-response and down-regulation of STAT1 before differentiation. This multi-trajectory analysis explores the cells' state transition with a multitude of perturbations in combination with traditional pathway analysis, leading to an encompassing picture of the dynamics of a therapeutically desired cell-state switching.

  16. The functional micro-organization of grid cells revealed by cellular-resolution imaging.

    PubMed

    Heys, James G; Rangarajan, Krsna V; Dombeck, Daniel A

    2014-12-03

    Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater microcircuit-level understanding of the brain's representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to nongrid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: the similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a "Mexican hat"-shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart.

  17. Organization of ventrolateral periolivary cells of the cat superior olive as revealed by PEP-19 immunocytochemistry and Nissl stain.

    PubMed

    Spirou, G A; Berrebi, A S

    1996-04-22

    Ventrolateral periolivary cell groups, through their descending projections to the cochlear nucleus (CN) and local projections to principal nuclei of the superior olive, may participate in brainstem mechanisms mediating such tasks as signal detection in noisy environments and sound localization. Understanding the function of these cell groups can be improved by increased knowledge of the organization of their synaptic inputs in relation to their cellular characteristics. Immunocytochemistry for PEP-19 (a putative calcium binding protein) reveals four patterns of immunolabeling within the ventrolateral periolivary region. Three of the patterns, which have distinct fiber and punctate labeling characteristics, help to define three subdivisions of the lateral nucleus of the trapezoid body (LNTB). The fourth pattern defines two other nuclei, the anterolateral periolivary nucleus (rostral) and the posterior periolivary nucleus (caudal), which display many immunoreactive cell bodies but little fiber and punctate labeling. One of the subdivisions of the LNTB contains large PEP-19 immunolabeled puncta arranged in pericellular nests. Analysis of Nissl-stained sections reveals a neuronal population that resembles globular cells of the ventral cochlear nucleus (VCN) and which colocalizes with pericellular nests of large immunolabeled puncta. Cell counts reveal that roughly 10,000 neurons constitute the cat ventrolateral periolivary region, 9,000 of which are found in the LNTB. Three-dimensional reconstructions of auditory brainstem nuclei clarify the complex spatial relationships among these structures.

  18. Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment

    PubMed Central

    2011-01-01

    Introduction The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised. Methods Here, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made. Results Several of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species. Conclusions These results provide new insights into genetic regulatory mechanisms of mammary development, particularly

  19. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer.

    PubMed

    Spike, Benjamin T; Engle, Dannielle D; Lin, Jennifer C; Cheung, Samantha K; La, Justin; Wahl, Geoffrey M

    2012-02-03

    Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate, and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and coexpress multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms, including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis, and therapy.

  20. Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

    PubMed Central

    Kaneko, Kumi; Umatani, Chie; Tadano, Hiroto; Ugajin, Atsushi; Nakaoka, Takayoshi; Paul, Rajib Kumar; Fujiyuki, Tomoko; Shirai, Kenichi; Kunieda, Takekazu; Takeuchi, Hideaki; Kubo, Takeo

    2013-01-01

    The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure. PMID:23990981

  1. Carbon-fiber microelectrode amperometry reveals sickle-cell-induced inflammation and chronic morphine effects on single mast cells.

    PubMed

    Manning, Benjamin M; Hebbel, Robert P; Gupta, Kalpna; Haynes, Christy L

    2012-03-16

    Sickle cell disease, caused by a mutation of hemoglobin, is characterized by a complex pathophysiology including an important inflammatory component. Mast cells are tissue-resident leukocytes known to influence a range of immune functions in a variety of different ways, largely through the secretion of biologically active mediators from preformed granules. However, it is not understood how mast cells influence the inflammatory environment in sickle cell disease. A notable consequence of sickle cell disease is severe pain. Therefore, morphine is often used to treat this disease. Because mast cells express opioid receptors, it is pertinent to understand how chronic morphine exposure influences mast cell function and inflammation in sickle cell disease. Herein, carbon-fiber microelectrode amperometry (CFMA) was used to monitor the secretion of immunoactive mediators from single mast cells. CFMA enabled the detection and quantification of discrete exocytotic events from single mast cells. Mast cells from two transgenic mouse models expressing human sickle hemoglobin (hBERK1 and BERK) and a control mouse expressing normal human hemoglobin (HbA-BERK) were monitored using CFMA to explore the impact of sickle-cell-induced inflammation and chronic morphine exposure on mast cell function. This work, utilizing the unique mechanistic perspective provided by CFMA, describes how mast cell function is significantly altered in hBERK1 and BERK mice, including decreased serotonin released compared to HbA-BERK controls. Furthermore, morphine was shown to significantly increase the serotonin released from HbA-BERK mast cells and demonstrated the capacity to reverse the observed sickle-cell-induced changes in mast cell function.

  2. A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.

    PubMed

    Wong, Michael Thomas; Ong, David Eng Hui; Lim, Frances Sheau Huei; Teng, Karen Wei Weng; McGovern, Naomi; Narayanan, Sriram; Ho, Wen Qi; Cerny, Daniela; Tan, Henry Kun Kiaang; Anicete, Rosslyn; Tan, Bien Keem; Lim, Tony Kiat Hon; Chan, Chung Yip; Cheow, Peng Chung; Lee, Ser Yee; Takano, Angela; Tan, Eng-Huat; Tam, John Kit Chung; Tan, Ern Yu; Chan, Jerry Kok Yen; Fink, Katja; Bertoletti, Antonio; Ginhoux, Florent; Curotto de Lafaille, Maria Alicia; Newell, Evan William

    2016-08-16

    Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.

  3. Cell autonomous defects in cortical development revealed by two-color chimera analysis.

    PubMed

    Kwiatkowski, Adam V; Garner, Craig C; Nelson, W James; Gertler, Frank B

    2009-05-01

    A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.

  4. Cells transplanted onto the surface of the glial scar reveal hidden potential for functional neural regeneration

    PubMed Central

    Sekiya, Tetsuji; Holley, Matthew C.; Hashido, Kento; Ono, Kazuya; Shimomura, Koichiro; Horie, Rie T.; Hamaguchi, Kiyomi; Yoshida, Atsuhiro; Sakamoto, Tatsunori; Ito, Juichi

    2015-01-01

    Cell transplantation therapy has long been investigated as a therapeutic intervention for neurodegenerative disorders, including spinal cord injury, Parkinson’s disease, and amyotrophic lateral sclerosis. Indeed, patients have high hopes for a cell-based therapy. However, there are numerous practical challenges for clinical translation. One major problem is that only very low numbers of donor cells survive and achieve functional integration into the host. Glial scar tissue in chronic neurodegenerative disorders strongly inhibits regeneration, and this inhibition must be overcome to accomplish successful cell transplantation. Intraneural cell transplantation is considered to be the best way to deliver cells to the host. We questioned this view with experiments in vivo on a rat glial scar model of the auditory system. Our results show that intraneural transplantation to the auditory nerve, preceded by chondroitinase ABC (ChABC)-treatment, is ineffective. There is no functional recovery, and almost all transplanted cells die within a few weeks. However, when donor cells are placed on the surface of a ChABC-treated gliotic auditory nerve, they autonomously migrate into it and recapitulate glia- and neuron-guided cell migration modes to repair the auditory pathway and recover auditory function. Surface transplantation may thus pave the way for improved functional integration of donor cells into host tissue, providing a less invasive approach to rescue clinically important neural tracts. PMID:26080415

  5. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    NASA Astrophysics Data System (ADS)

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-10-01

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

  6. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    PubMed Central

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-01-01

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines. PMID:26489853

  7. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.

    PubMed

    Maass, Katie F; Kulkarni, Chethana; Quadir, Mohiuddin A; Hammond, Paula T; Betts, Alison M; Wittrup, Karl Dane

    2015-12-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.

  8. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    SciTech Connect

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; Xia, Yankai

    2015-10-22

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cell cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Furthermore, glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.

  9. Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.

    PubMed

    Sheng, X Rebecca; Matunis, Erika

    2011-08-01

    Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.

  10. Autonomy and Non-autonomy of Angiogenic Cell Movements Revealed by Experiment-Driven Mathematical Modeling.

    PubMed

    Sugihara, Kei; Nishiyama, Koichi; Fukuhara, Shigetomo; Uemura, Akiyoshi; Arima, Satoshi; Kobayashi, Ryo; Köhn-Luque, Alvaro; Mochizuki, Naoki; Suda, Toshio; Ogawa, Hisao; Kurihara, Hiroki

    2015-12-01

    Angiogenesis is a multicellular phenomenon driven by morphogenetic cell movements. We recently reported morphogenetic vascular endothelial cell (EC) behaviors to be dynamic and complex. However, the principal mechanisms orchestrating individual EC movements in angiogenic morphogenesis remain largely unknown. Here we present an experiment-driven mathematical model that enables us to systematically dissect cellular mechanisms in branch elongation. We found that cell-autonomous and coordinated actions governed these multicellular behaviors, and a cell-autonomous process sufficiently illustrated essential features of the morphogenetic EC dynamics at both the single-cell and cell-population levels. Through refining our model and experimental verification, we further identified a coordinated mode of tip EC behaviors regulated via a spatial relationship between tip and follower ECs, which facilitates the forward motility of tip ECs. These findings provide insights that enhance our mechanistic understanding of not only angiogenic morphogenesis, but also other types of multicellular phenomenon.

  11. Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

    PubMed

    Shimada, Kenichi; Skouta, Rachid; Kaplan, Anna; Yang, Wan Seok; Hayano, Miki; Dixon, Scott J; Brown, Lewis M; Valenzuela, Carlos A; Wolpaw, Adam J; Stockwell, Brent R

    2016-07-01

    Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

  12. Live imaging reveals the progenitors and cell dynamics of limb regeneration

    PubMed Central

    Alwes, Frederike; Enjolras, Camille; Averof, Michalis

    2016-01-01

    Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg. DOI: http://dx.doi.org/10.7554/eLife.19766.001 PMID:27776632

  13. Single neuron transcriptome analysis can reveal more than cell type classification

    PubMed Central

    Harbom, Lise J.; Chronister, William D.

    2016-01-01

    A recent single cell mRNA sequencing study by Dueck et al. compares neuronal transcriptomes to the transcriptomes of adipocytes and cardiomyocytes. Single cell ‘omic approaches such as those used by the authors are at the leading edge of molecular and biophysical measurement. Many groups are currently employing single cell sequencing approaches to understand cellular heterogeneity in cancer and during normal development. These single cell approaches also are beginning to address long‐standing questions regarding nervous system diversity. Beyond an innate interest in cataloging cell type diversity in the brain, single cell neuronal diversity has important implications for neurotypic neural circuit function and for neurological disease. Herein, we review the authors’ methods and findings, which most notably include evidence of unique expression profiles in some single neurons. PMID:26749010

  14. Scanning Angle Interference Microscopy Reveals Cell Dynamics at the Nano-scale

    PubMed Central

    Paszek, Matthew J.; DuFort, Christopher C.; Rubashkin, Matthew G.; Davidson, Mike W.; Thorn, Kurt S.; Liphardt, Jan T.; Weaver, Valerie M.

    2012-01-01

    Emerging questions in cell biology necessitate nanometer-scale imaging in live cells. Here we present scanning angle interference microscopy, capable of localizing fluorescent objects with nanometer-scale precision along the optical axis in motile cellular structures. We use this approach to resolve nano-topographical features of the cell membrane and cytoskeleton, as well as the temporal evolution, three-dimensional architecture, and nano-scale dynamics of focal adhesion complexes. PMID:22751201

  15. Transcript Analysis Reveals a Specific HOX Signature Associated with Positional Identity of Human Endothelial Cells

    PubMed Central

    Toshner, Mark; Dunmore, Benjamin J.; McKinney, Eoin F.; Southwood, Mark; Caruso, Paola; Upton, Paul D.; Waters, John P.; Ormiston, Mark L.; Skepper, Jeremy N.; Nash, Gerard; Rana, Amer A.; Morrell, Nicholas W.

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole “HOX transcriptome” of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity. PMID:24651450

  16. Transcript analysis reveals a specific HOX signature associated with positional identity of human endothelial cells.

    PubMed

    Toshner, Mark; Dunmore, Benjamin J; McKinney, Eoin F; Southwood, Mark; Caruso, Paola; Upton, Paul D; Waters, John P; Ormiston, Mark L; Skepper, Jeremy N; Nash, Gerard; Rana, Amer A; Morrell, Nicholas W

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole "HOX transcriptome" of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity.

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

    PubMed

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

    2015-05-07

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

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

    PubMed Central

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

    2015-01-01

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy. PMID:25950479

  19. Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory.

    PubMed

    Oliveira, Giacomo; Ruggiero, Eliana; Stanghellini, Maria Teresa Lupo; Cieri, Nicoletta; D'Agostino, Mattia; D'Agostino, Mattio; Fronza, Raffaele; Lulay, Christina; Dionisio, Francesca; Mastaglio, Sara; Greco, Raffaella; Peccatori, Jacopo; Aiuti, Alessandro; Ambrosi, Alessandro; Biasco, Luca; Bondanza, Attilio; Lambiase, Antonio; Traversari, Catia; Vago, Luca; von Kalle, Christof; Schmidt, Manfred; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

    2015-12-09

    Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK(+) cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)- and Flu-specific TK(+) cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRβ clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

  20. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    DOE PAGES

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; ...

    2015-10-22

    Thirdhand smoke (THS) is a new term for the toxins in cigarette smoke that linger in the environment long after the cigarettes are extinguished. The effects of THS exposure on male reproduction have not yet been studied. In this study, metabolic changes in male germ cell lines (GC-2 and TM-4) were analyzed after THS treatment for 24 h. THS-loaded chromatography paper samples were generated in a laboratory chamber system and extracted in DMEM. At a paper: DMEM ratio of 50 μg/ml, cell viability in both cell lines was normal, as measured by the MTT assay and markers of cytotoxicity, cellmore » cycle, apoptosis and ROS production were normal as measured by quantitative immunofluorescence. Metabolomic analysis was performed on methanol extracts of GC-2 and TM-4 cells. Furthermore, glutathione metabolism in GC-2 cells, and nucleic acid and ammonia metabolism in TM-4 cells, was changed significantly by THS treatment. RT-PCR analyses of mRNA for enzyme genes Gss and Ggt in GC-2 cells, and TK, SMS and Glna in TM-4 cells reinforced these findings, showing changes in the levels of enzymes involved in the relevant pathways. In conclusion, exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell lines.« less

  1. Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri

    NASA Astrophysics Data System (ADS)

    Qi, Zhenhua; Pei, Guangsheng; Chen, Lei; Zhang, Weiwen

    2014-12-01

    Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

  2. Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri.

    PubMed

    Qi, Zhenhua; Pei, Guangsheng; Chen, Lei; Zhang, Weiwen

    2014-12-15

    Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

  3. Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex.

    PubMed

    Brault, Jean-Baptiste; Khou, Cécile; Basset, Justine; Coquand, Laure; Fraisier, Vincent; Frenkiel, Marie-Pascale; Goud, Bruno; Manuguerra, Jean-Claude; Pardigon, Nathalie; Baffet, Alexandre D

    2016-08-01

    The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

  4. Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq

    SciTech Connect

    Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; Gaffrey, Matthew J.; Hu, Dehong; Szymanski, Craig J.; Xie, Yumei; Melby, Eric S.; Dohnalkova, Alice; Taylor, Ronald C.; Grate, Eva K.; Cooley, Scott K.; McDermott, Jason E.; Heredia-Langner, Alejandro; Orr, Galya

    2016-11-22

    The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. Here we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with upregulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly downregulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong upregulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis and organelle activities. In contrast, strategies unique to carboxylated QDs showed upregulation of DNA repair and RNA activities, and decreased regulation of cell division, coupled in some cases with upregulation of stress responses and ATP related functions. Together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified, proactive defenses or repairs of the NP insults.

  5. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived HGF and fibronectin in angiogenesis

    PubMed Central

    Newman, Andrew C.; Chou, Wayne; Welch-Reardon, Katrina M.; Fong, Ashley H.; Popson, Stephanie A.; Phan, Duc Thien; Sandoval, Daniel R.; Nguyen, Dananh P.; Gershon, Paul D.; Hughes, Christopher C. W.

    2013-01-01

    Objective Angiogenesis requires tightly coordinated cross-talk between endothelial cells and stromal cells such as fibroblasts and smooth muscle cells. The specific molecular mechanisms moderating this process are still poorly understood. Method and Results Stromal cell-derived factors are essential for endothelial cell sprouting and lumen formation. We therefore compared the abilities of two primary fibroblast isolates and a primary smooth muscle cell isolate to promote in vitro angiogenesis and analyzed their secretomes using a combination of nanoLC-MS/MS, qPCR and ELISA. Each isolate exhibited a different level of angiogenic ability. Using quantitative MS, we then compared the secretomes of a fibroblast isolate exhibiting low angiogenic activity, a fibroblast isolate exhibiting high angiogenic activity and human umbilical vein endothelial cells. High angiogenic fibroblast supernatants exhibited an over-abundance of proteins associated with extracellular matrix constituents compared to low angiogenic fibroblasts or endothelial cells. Finally, siRNA technology and purified protein were used to confirm a role for stromal cell-derived hepatocyte growth factor and fibronectin in inducing endothelial cell sprouting. Conclusion Differences in stromal cell ability to induce angiogenesis are due to differences in the secreted proteomes of both extracellular matrix proteins and pro-angiogenic growth factors. PMID:23288153

  6. Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity.

    PubMed

    Chapman, Matthew R; Balakrishnan, Karthik R; Li, Ju; Conboy, Michael J; Huang, Haiyan; Mohanty, Swomitra K; Jabart, Eric; Hack, James; Conboy, Irina M; Sohn, Lydia L

    2013-04-01

    Traditional cell-screening techniques such as FACS and MACS are better suited for large numbers of cells isolated from bulk tissue and cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, these techniques rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. To address these challenges, we have developed a novel, label-free stem-cell analysis and sorting platform capable of quantifying cell-surface marker expression of single functional organ stem cells directly isolated from their micro-anatomical niche. Using our unique platform, we have discovered a remarkable heterogeneity in both the regenerative capacity and expression of CXCR4, β1-integrin, Sca-1, M-cadherin, Syndecan-4, and Notch-1 in freshly isolated muscle stem (satellite) cells residing on different, single myofibers and have identified a small population of Sca-1(+)/Myf5(+) myogenic satellite cells. Our results demonstrate the utility of our single-cell platform for uncovering and functionally characterizing stem-cell heterogeneity in the organ microniche.

  7. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

    PubMed

    Zeisel, Amit; Muñoz-Manchado, Ana B; Codeluppi, Simone; Lönnerberg, Peter; La Manno, Gioele; Juréus, Anna; Marques, Sueli; Munguba, Hermany; He, Liqun; Betsholtz, Christer; Rolny, Charlotte; Castelo-Branco, Gonçalo; Hjerling-Leffler, Jens; Linnarsson, Sten

    2015-03-06

    The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

  8. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    PubMed

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  9. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    PubMed Central

    Noutsi, Pakiza; Gratton, Enrico; Chaieb, Sahraoui

    2016-01-01

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines. PMID:27362860

  10. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    SciTech Connect

    Carmen Herranz, Ma; Mingarro, Ismael; Pallas, Vicente . E-mail: vpallas@ibmcp.upv.es

    2005-08-15

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

  11. Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

    PubMed

    Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

    2012-08-08

    Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

  12. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  13. Rhodamine123 reveals heterogeneity within murine Lin-, Sca-1+ hemopoietic stem cells

    PubMed Central

    1992-01-01

    Murine bone marrow Lin-, Ly6A/E+ cells have been fractionated on the basis of rhodamine123 retention into Rh123med/hi and Rh123lo subpopulations. These populations have different responses to hemopoietic growth factors with respect to in vitro colony formation. Cells from either fraction were not stimulated by only granulocyte- colony-stimulating factor (G-CSF), granulocyte/macrophage colony- stimulating factor (GM-CSF), macrophage colony-stimulating factor (M- CSF), interleukins 1 and 6 (IL-1 and -6), or leukemia inhibitory factor (LIF) alone. The Rh123med/hi, but not the Rh123lo fraction, contained cells that could be stimulated by either stem cell factor (SCF) or IL-3 alone. When combinations of growth factors were added, the Rh123med/hi fraction produced more colonies, and responded to a wider range of factor combinations than the Rh123lo population. When tested in vivo, both populations contained no detectable day 8 colony-forming unit- spleen (CFU-S), and similar frequencies of day 13 CFU-S. When transplanted into lethally irradiated recipients (100 cells/recipient), significant numbers of donor cells (67-73%) were found in the peripheral blood of Rh123lo recipients. Both myeloid and lymphoid cells were of donor origin. By comparison, the Rh123med/hi population produced recipients with 1-2% donor cells in peripheral blood, the majority of which were lymphoid. PMID:1375260

  14. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability.

    PubMed

    Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M; Dianov, Grigory L

    2015-04-20

    Genetic instability, provoked by exogenous mutagens, is well linked to initiation of cancer. However, even in unstressed cells, DNA undergoes a plethora of spontaneous alterations provoked by its inherent chemical instability and the intracellular milieu. Base excision repair (BER) is the major cellular pathway responsible for repair of these lesions, and as deficiency in BER activity results in DNA damage it has been proposed that it may trigger the development of sporadic cancers. Nevertheless, experimental evidence for this model remains inconsistent and elusive. Here, we performed a proteomic analysis of BER deficient human cells using stable isotope labelling with amino acids in cell culture (SILAC), and demonstrate that BER deficiency, which induces genetic instability, results in dramatic changes in gene expression, resembling changes found in many cancers. We observed profound alterations in tissue homeostasis, serine biosynthesis, and one-carbon- and amino acid metabolism, all of which have been identified as cancer cell 'hallmarks'. For the first time, this study describes gene expression changes characteristic for cells deficient in repair of endogenous DNA lesions by BER. These expression changes resemble those observed in cancer cells, suggesting that genetically unstable BER deficient cells may be a source of pre-cancerous cells.

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

    PubMed

    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

    2016-02-15

    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.

  16. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    PubMed Central

    Villar, Rina F.; Patel, Jinal; Weaver, Grant C.; Kanekiyo, Masaru; Wheatley, Adam K.; Yassine, Hadi M.; Costello, Catherine E.; Chandler, Kevin B.; McTamney, Patrick. M.; Nabel, Gary J.; McDermott, Adrian B.; Mascola, John R.; Carr, Steven A.; Lingwood, Daniel

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed. PMID:27796362

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

    PubMed Central

    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

  18. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death

    PubMed Central

    Furness, SGB; Hare, DL; Kourakis, A; Turnley, AM; Wookey, PJ

    2016-01-01

    We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis. PMID:27777788

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

    PubMed

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

    2013-03-01

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

  20. Transcriptional analysis of histone deacetylase family members reveal similarities between differentiating and aging spermatogonial stem cells.

    PubMed

    Kofman, Amber E; Huszar, Jessica M; Payne, Christopher J

    2013-02-01

    The differentiation of adult stem cells involves extensive chromatin remodeling, mediated in part by the gene products of histone deacetylase (HDAC) family members. While the transcriptional downregulation of HDACs can impede stem cell self-renewal in certain contexts, it may also promote stem cell maintenance under other circumstances. In self-renewing, differentiating, and aging spermatogonial stem cells (SSCs), the gene expression dynamics of HDACs have not yet been characterized. To gain further insight with these studies, we analyzed the transcriptional profiles of six HDAC family members, previously identified to be the most highly expressed in self-renewing SSCs, during stem cell differentiation and aging. Here we discovered that in both differentiating and aging SSCs the expression of Sirt4 increases, while the expression of Hdac2, Hdac6, and Sirt1 decreases. When SSCs are exposed to the lifespan-enhancing drug rapamycin in vivo, the resultant HDAC gene expression patterns are opposite of those seen in the differentiating and aging SSCs, with increased Hdac2, Hdac6, and Sirt1 and decreased Hdac8, Hdac9, and Sirt4. Our findings suggest that HDACs important for stem cell maintenance and oxidative capacity are downregulated as adult stem cells differentiate or age. These results provide important insights into the epigenetic regulation of stem cell differentiation and aging in mammals.

  1. Molecular dissection of zyxin function reveals its involvement in cell motility.

    PubMed

    Drees, B E; Andrews, K M; Beckerle, M C

    1999-12-27

    Spatially controlled actin filament assembly is critical for numerous processes, including the vectorial cell migration required for wound healing, cell- mediated immunity, and embryogenesis. One protein implicated in the regulation of actin assembly is zyxin, a protein concentrated at sites where the fast growing ends of actin filaments are enriched. To evaluate the role of zyxin in vivo, we developed a specific peptide inhibitor of zyxin function that blocks its interaction with alpha-actinin and displaces it from its normal subcellular location. Mislocalization of zyxin perturbs cell migration and spreading, and affects the behavior of the cell edge, a structure maintained by assembly of actin at sites proximal to the plasma membrane. These results support a role for zyxin in cell motility, and demonstrate that the correct positioning of zyxin within the cell is critical for its physiological function. Interestingly, the mislocalization of zyxin in the peptide-injected cells is accompanied by disturbances in the distribution of Ena/VASP family members, proteins that have a well-established role in promoting actin assembly. In concert with previous work, our findings suggest that zyxin promotes the spatially restricted assembly of protein complexes necessary for cell motility.

  2. Receptor binding and cell entry of Old World arenaviruses reveal novel aspects of virus-host interaction.

    PubMed

    Kunz, Stefan

    2009-05-10

    Ten years ago, the first cellular receptor for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the highly pathogenic Lassa virus (LASV) was identified as alpha-dystroglycan (alpha-DG), a versatile receptor for proteins of the extracellular matrix (ECM). Biochemical analysis of the interaction of alpha-DG with arenaviruses and ECM proteins revealed a strikingly similar mechanism of receptor recognition that critically depends on specific sugar modification on alpha-DG involving a novel class of putative glycosyltransferase, the LARGE proteins. Interestingly, recent genome-wide detection and characterization of positive selection in human populations revealed evidence for positive selection of a locus within the LARGE gene in populations from Western Africa, where LASV is endemic. While most enveloped viruses that enter the host cell in a pH-dependent manner use clathrin-mediated endocytosis, recent studies revealed that the Old World arenaviruses LCMV and LASV enter the host cell predominantly via a novel and unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the virus is rapidly delivered to endosomes via an unusual route of vesicular trafficking that is largely independent of the small GTPases Rab5 and Rab7. Since infection of cells with LCMV and LASV depends on DG, this unusual endocytotic pathway could be related to normal cellular trafficking of the DG complex. Alternatively, engagement of arenavirus particles may target DG for an endocytotic pathway not normally used in uninfected cells thereby inducing an entry route specifically tailored to the pathogen's needs.

  3. A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation1

    PubMed Central

    Wang, Hongsheng; Yan, Ming; Sun, Jiafang; Jain, Shweta; Yoshimi, Ryusuke; Abolfath, Sanaz Momben; Ozato, Keiko; Coleman, William G.; Ng, Ashley P.; Metcalf, Donald; DiRago, Ladina; Nutt, Stephen L.; Morse, Herbert C.

    2014-01-01

    The interferon regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. Here we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors (GMPs) and the common lymphoid progenitors (CLPs) but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous GMPs with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFPhi monocytic progenitors or EGFPlo granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell-committed (EGFPint) from those that are non-committed. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors. PMID:25024380

  4. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    SciTech Connect

    Vancini, Ricardo; Kramer, Laura D.; Ribeiro, Mariana; Hernandez, Raquel; Brown, Dennis

    2013-01-20

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  5. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.

    PubMed

    Petropoulos, Sophie; Edsgärd, Daniel; Reinius, Björn; Deng, Qiaolin; Panula, Sarita Pauliina; Codeluppi, Simone; Plaza Reyes, Alvaro; Linnarsson, Sten; Sandberg, Rickard; Lanner, Fredrik

    2016-05-05

    Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

  6. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians

    PubMed Central

    Forsthoefel, David J.; James, Noelle P.; Escobar, David J.; Stary, Joel M.; Vieira, Ana P.; Waters, Forrest A.; Newmark, Phillip A.

    2012-01-01

    SUMMARY Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of post-mitotic tissues. Understanding how these processes are orchestrated requires characterizing cell type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis, and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling post-embryonic organogenesis. PMID:23079596

  7. High-Throughput Proteomics Reveals the Unicellular Roots of Animal Phosphosignaling and Cell Differentiation.

    PubMed

    Sebé-Pedrós, Arnau; Peña, Marcia Ivonne; Capella-Gutiérrez, Salvador; Antó, Meritxell; Gabaldón, Toni; Ruiz-Trillo, Iñaki; Sabidó, Eduard

    2016-10-24

    Cell-specific regulation of protein levels and activity is essential for the distribution of functions among multiple cell types in animals. The finding that many genes involved in these regulatory processes have a premetazoan origin raises the intriguing possibility that the mechanisms required for spatially regulated cell differentiation evolved prior to the appearance of animals. Here, we use high-throughput proteomics in Capsaspora owczarzaki, a close unicellular relative of animals, to characterize the dynamic proteome and phosphoproteome profiles of three temporally distinct cell types in this premetazoan species. We show that life-cycle transitions are linked to extensive proteome and phosphoproteome remodeling and that they affect key genes involved in animal multicellularity, such as transcription factors and tyrosine kinases. The observation of shared features between Capsaspora and metazoans indicates that elaborate and conserved phosphosignaling and proteome regulation supported temporal cell-type differentiation in the unicellular ancestor of animals.

  8. An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians.

    PubMed

    Forsthoefel, David J; James, Noëlle P; Escobar, David J; Stary, Joel M; Vieira, Ana P; Waters, Forrest A; Newmark, Phillip A

    2012-10-16

    Planarians grow and regenerate organs by coordinating proliferation and differentiation of pluripotent stem cells with remodeling of postmitotic tissues. Understanding how these processes are orchestrated requires characterizing cell-type-specific gene expression programs and their regulation during regeneration and homeostasis. To this end, we analyzed the expression profile of planarian intestinal phagocytes, cells responsible for digestion and nutrient storage/distribution. Utilizing RNA interference, we identified cytoskeletal regulators required for intestinal branching morphogenesis and a modulator of bioactive sphingolipid metabolism, ceramide synthase, required for the production of functional phagocytes. Additionally, we found that a gut-enriched homeobox transcription factor, nkx-2.2, is required for somatic stem cell proliferation, suggesting a niche-like role for phagocytes. Identification of evolutionarily conserved regulators of intestinal branching, differentiation, and stem cell dynamics demonstrates the utility of the planarian digestive system as a model for elucidating the mechanisms controlling postembryonic organogenesis.

  9. Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

    PubMed Central

    Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

    2016-01-01

    ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA

  10. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    PubMed

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

  11. Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals.

    PubMed

    Konadu, Kateena Addae; Huang, Ming Bo; Roth, William; Armstrong, Wendy; Powell, Michael; Villinger, Francois; Bond, Vincent

    2016-01-05

    Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

  12. Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensin-receptor-1-positive tumor targeting

    PubMed Central

    Jia, Yinnong; Shi, Wen; Zhou, Zhengyuan; Wagh, Nilesh K.; Fan, Wei; Brusnahan, Susan K.; Garrison, Jered C.

    2015-01-01

    Introduction Neurotensin receptor 1 (NTR1) is overexpressed in many cancers types. Neurotensin (NT), a 13 amino acid peptide, is the native ligand for NTR1 and exhibits high (nM) affinity to the receptor. Many laboratories have been investigating the development of diagnostic and therapeutic radiopharmaceuticals for NTR1-positive cancers based on the NT peptide. To improve the biological performance for targeting NTR1, we proposed NT analogs with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers. Methods We synthesized four NTR1-targeted conjugates with spacer lengths from 0 to 9 atoms (null (N0), β-Ala-OH (N1), 5-Ava-OH (N2), and 8-Aoc-OH (N3)) between the DOTA and the pharmacophore. In vitro competitive binding, internalization and efflux studies were performed on all four NT analogs. Based on these findings, metabolism studies were carried out on our best performing conjugate, 177Lu-N1. Lastly, in vivo biodistribution and SPECT/CT imaging studies were performed using 177Lu-N1 in an HT-29 xenograft mouse model. Results As shown in competitive binding assay, the NT analogs with different spacers (N1, N2 and N3) exhibited lower IC50 values than the NT analog without a spacer (N0). Furthermore, N1 revealed higher retention in HT-29 cells with more rapid internalization and slower efflux than the other NT analogs. In vivo biodistribution and SPECT/CT imaging studies of 177Lu-N1 demonstrated excellent accumulation (3.1 ± 0.4 %ID/g) in the NTR1-positive tumors at 4 h post-administration. Conclusions The DOTA chelation system demonstrated some modest steric inhibition of the pharmacophore. However, the insertion of a 4-atom hydrocarbon spacer group restored optimal binding affinity of the analog. The in vivo assays indicated that 177Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors. PMID:26302836

  13. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    PubMed

    Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

    2010-10-14

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  14. Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity.

    PubMed

    Small, Eliza C; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan; Licht, Jonathan D

    2014-06-17

    Nucleosomes, the basic unit of chromatin, have a critical role in the control of gene expression. Nucleosome positions have generally been determined by examining bulk populations of cells and then correlated with overall gene expression. Here, we describe a technique to determine nucleosome positioning in single cells by virtue of the ability of the nucleosome to protect DNA from GpC methylation. In the acid phosphatase inducible PHO5 gene, we find that there is significant cell-to-cell variation in nucleosome positions and shifts in nucleosome positioning correlate with changes in gene expression. However, nucleosome positioning is not absolute, and even with major shifts in gene expression, some cells fail to change nucleosome configuration. Mutations of the PHO5 promoter that introduce a poly(dA:dT) tract-stimulated gene expression under nonpermissive conditions led to shifts of positioned nucleosomes similar to induction of PHO5. By contrast, mutations that altered AA/TT/AT periodicity reduced gene expression upon PHO5 induction and stabilized nucleosomes in most cells, suggesting that enhanced nucleosome affinity for DNA antagonizes chromatin remodelers. Finally, we determined nucleosome positioning in two regions described as "fuzzy" or nucleosome-free when examined in a bulk assay. These regions consisted of distinct nucleosomes with a larger footprint for potential location and an increase population of cells lacking a nucleosome altogether. These data indicate an underlying complexity of nucleosome positioning that may contribute to the flexibility and heterogeneity of gene expression.

  15. Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics

    PubMed Central

    Quinn, T. Alexander; Camelliti, Patrizia; Rog-Zielinska, Eva A.; Siedlecka, Urszula; Poggioli, Tommaso; O'Toole, Eileen T.; Knöpfel, Thomas; Kohl, Peter

    2016-01-01

    Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte–nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart. PMID:27930302

  16. A simple force-motion relation for migrating cells revealed by multipole analysis of traction stress.

    PubMed

    Tanimoto, Hirokazu; Sano, Masaki

    2014-01-07

    For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dynamics of migrating cells. We measured the traction stress of Dictyostelium discoideum cells and investigated the lowest two moments, the force dipole and quadrupole moments, which reflect rotational and front-rear asymmetries of the stress field. We derived a simple force-motion relation in which cells migrate along the force dipole axis with a direction determined by the force quadrupole. Furthermore, as a complementary approach, we also investigated fine structures in the stress field that show front-rear asymmetric kinetics consistent with the multipole analysis. The tight force-motion relation enables us to predict cell migration only from the traction stress patterns.

  17. Effects of paclitaxel on EGFR endocytic trafficking revealed using quantum dot tracking in single cells.

    PubMed

    Li, Hui; Duan, Zhao-Wen; Xie, Ping; Liu, Yu-Ru; Wang, Wei-Chi; Dou, Shuo-Xing; Wang, Peng-Ye

    2012-01-01

    Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introduced to quantitatively study the dynamics of endocytic trafficking. Compared with the control cells, the velocity of directed motion was reduced by 30% due to the suppression of high speed movements of EGF-QDs along the microtubules in PTX-treated cells. The endocytic trafficking in PTX-treated cells was mainly via super-diffusive mode of motion, whereas in control cells, it was mostly via sub-diffusive mode of motion. Moreover, PTX shortened endosomal trafficking and prevented EGF-QDs from moving to the perinuclear area via the rapid delivery of EGF-QDs into the peripheral lysosomes. The present study may shed light on the mechanism of the effect of PTX on the treatment of lung cancer.

  18. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    PubMed Central

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  19. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival

    PubMed Central

    Kawamura, Tatsuro; Kawatani, Makoto; Muroi, Makoto; Kondoh, Yasumitsu; Futamura, Yushi; Aono, Harumi; Tanaka, Miho; Honda, Kaori; Osada, Hiroyuki

    2016-01-01

    Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor–induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival. PMID:27210421

  20. Superresolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth.

    PubMed

    Ishitsuka, Yuji; Savage, Natasha; Li, Yiming; Bergs, Anna; Grün, Nathalie; Kohler, Daria; Donnelly, Rebecca; Nienhaus, G Ulrich; Fischer, Reinhard; Takeshita, Norio

    2015-11-01

    Polar (directional) cell growth, a key cellular mechanism shared among a wide range of species, relies on targeted insertion of new material at specific locations of the plasma membrane. How these cell polarity sites are stably maintained during massive membrane insertion has remained elusive. Conventional live-cell optical microscopy fails to visualize polarity site formation in the crowded cell membrane environment because of its limited resolution. We have used advanced live-cell imaging techniques to directly observe the localization, assembly, and disassembly processes of cell polarity sites with high spatiotemporal resolution in a rapidly growing filamentous fungus, Aspergillus nidulans. We show that the membrane-associated polarity site marker TeaR is transported on microtubules along with secretory vesicles and forms a protein cluster at that point of the apical membrane where the plus end of the microtubule touches. There, a small patch of membrane is added through exocytosis, and the TeaR cluster gets quickly dispersed over the membrane. There is an incessant disassembly and reassembly of polarity sites at the growth zone, and each new polarity site locus is slightly offset from preceding ones. On the basis of our imaging results and computational modeling, we propose a transient polarity model that explains how cell polarity is stably maintained during highly active directional growth.

  1. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    PubMed

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  2. The functional micro-organization of grid cells revealed by cellular-resolution imaging

    PubMed Central

    Heys, James G.; Rangarajan, Krsna V.; Dombeck, Daniel A.

    2015-01-01

    Summary Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater micro-circuit level understanding of the brain’s representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to non-grid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: The similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a “Mexican Hat” shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart. PMID:25467986

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

    PubMed Central

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

    2014-01-01

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

  4. Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen

    PubMed Central

    Lambert Emo, Kris; Hyun, Young-min; Barilla, Christopher; Gerber, Scott; Fowell, Deborah; Kim, Minsoo

    2016-01-01

    During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8–10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection. PMID:27644089

  5. Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions

    PubMed Central

    Fischer, Adrian; Warscheid, Bettina; Weber, Wilfried; Radziwill, Gerald

    2016-01-01

    Scaffold proteins such as the multidomain protein CNK1 orchestrate the signalling network by integrating and controlling the underlying pathways. Using an optogenetic approach to stimulate CNK1 uncoupled from upstream effectors, we identified selective clusters of CNK1 that either stimulate RAF-MEK-ERK or AKT signalling depending on the light intensity applied. OptoCNK1 implemented in MCF7 cells induces differentiation at low light intensity stimulating ERK activity whereas stimulation of AKT signalling by higher light intensity promotes cell proliferation. CNK1 clustering in response to increasing EGF concentrations revealed that CNK1 binds to RAF correlating with ERK activation at low EGF dose. At higher EGF dose active AKT binds to CNK1 and phosphorylates and inhibits RAF. Knockdown of CNK1 protects CNK1 from this AKT/RAF crosstalk. In C2 skeletal muscle cells CNK1 expression is induced with the onset of differentiation. Hence, AKT-bound CNK1 counteracts ERK stimulation in differentiated but not in proliferating cells. Ectopically expressed CNK1 facilitates C2 cell differentiation and knockdown of CNK1 impaired the transcriptional network underlying C2 cell differentiation. Thus, CNK1 expression, CNK1 clustering and the thereto related differential signalling processes decide on proliferation and differentiation in a cell type- and cell stage-dependent manner by orchestrating AKT and RAF signalling. PMID:27901111

  6. Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

    PubMed

    Hardy, W Reef; Moldovan, Nicanor I; Moldovan, Leni; Livak, Kenneth J; Datta, Krishna; Goswami, Chirayu; Corselli, Mirko; Traktuev, Dmitry O; Murray, Iain R; Péault, Bruno; March, Keith

    2017-02-24

    Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31(-) /CD45(-) /CD34(+) /CD146(-) cells (adventitial stromal/stem cells [ASCs]) and CD31(-) /CD45(-) /CD34(-) /CD146(+) cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDH(br) ASC (most primitive); (b) ALDH(dim) ASC; (c) ALDH(br) PC; (d) ALDH(dim) PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and

  7. Lipid Profiling Reveals Arachidonate Deficiency in RAW264.7 Cells: Structural and Functional Implications†

    PubMed Central

    Rouzer, Carol A.; Ivanova, Pavlina T.; Byrne, Mark O.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex

    2008-01-01

    Glycerophospholipids containing arachidonic acid (20:4) serve as the precursors for an array of biologically active lipid mediators, most of which are produced by macrophages. We have applied mass spectrometry-based lipid profiling technology to evaluate the glycerophospholipid structure and composition of two macrophage populations, resident peritoneal macrophages and RAW264.7 cells, with regard to their potential for 20:4-based lipid mediator biosynthesis. Fatty acid analysis indicated that RAW264.7 cells were deficient in 20:4 (10 ± 1 mole percent) as compared to peritoneal macrophages (26 ± 1 mole percent). Mass spectrometry of total glycerophospholipids demonstrated a marked difference in the distribution of lipid species, including reduced levels of 20:4-containing lipids, in RAW264.7 cells as compared to peritoneal macrophages. Enrichment of RAW264.7 cells with 20:4 increased the fatty acid to 20 ± 1 mole percent. However, the distribution of the incorporated 20:4 remained different from that of peritoneal macrophages. RAW264.7 cells pretreated with granulocyte-macrophage colony stimulating factor followed by lipopolysaccharide and interferon-gamma mobilized similar quantities of 20:4 and produced similar amounts of prostaglandins as peritoneal macrophages treated with LPS alone. LPS treatment resulted in detectable changes in specific 20:4-containing glycerophospholipids in peritoneal cells, but not in RAW264.7 cells. 20:4-enriched RAW264.7 cells lost 88% of the incorporated fatty acid during the LPS incubation without additional prostaglandin synthesis. These results illustrate that large differences in glycerophospholipid composition may exist, even in closely related cell populations, and demonstrate the importance of interpreting the potential for lipid-mediator biosynthesis in the context of overall glycerophospholipid composition. PMID:17144673

  8. Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation.

    PubMed

    Solaimani Kartalaei, Parham; Yamada-Inagawa, Tomoko; Vink, Chris S; de Pater, Emma; van der Linden, Reinier; Marks-Bluth, Jonathon; van der Sloot, Anthon; van den Hout, Mirjam; Yokomizo, Tomomasa; van Schaick-Solernó, M Lucila; Delwel, Ruud; Pimanda, John E; van IJcken, Wilfred F J; Dzierzak, Elaine

    2015-01-12

    Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.

  9. Suppression of Nestin reveals a critical role for p38-EGFR pathway in neural progenitor cell proliferation

    PubMed Central

    Hu, Wentao; Lu, Hong; Wang, Shang; Yin, Wenhan; Liu, Xujie; Dong, Lin; Chiu, Richard; Shen, Li; Lu, Wen-Jing; Lan, Feng

    2016-01-01

    The expression of intermediate filament Nestin is necessary for the neural progenitor cells (NPCs) to maintain stemness, but the underlying cellular and molecular mechanism remains unclear. In this study, we demonstrated that Nestin is required for the self-renew of NPCs through activating MAPK and EGFR pathways. Knockdown of Nestin by shRNA inhibited cell cycle progression and proliferation in mouse NPCs. Moreover, suppression of Nestin reduced expression of the epidermal growth factor receptor (EGFR) in NPCs and inhibited the mitogenic effects of EGF on these cells. Treatment of NPCs with p38-MAPK inhibitor PD169316 reversed cell cycle arrest caused by the knockdown of Nestin. Our findings indicate that Nestin promotes NPC proliferation via p38-MAPK and EGFR pathways, and reveals the necessity of these pathways in NPCs self-renewal. PMID:27894083

  10. Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation

    PubMed Central

    Solaimani Kartalaei, Parham; Yamada-Inagawa, Tomoko; Vink, Chris S.; de Pater, Emma; van der Linden, Reinier; Marks-Bluth, Jonathon; van der Sloot, Anthon; van den Hout, Mirjam; Yokomizo, Tomomasa; van Schaick-Solernó, M. Lucila; Delwel, Ruud; Pimanda, John E.; van IJcken, Wilfred F.J.

    2015-01-01

    Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the “heptad” complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs. PMID:25547674

  11. Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy.

    PubMed

    Hughes, Louise; Borrett, Samantha; Towers, Katie; Starborg, Tobias; Vaughan, Sue

    2017-02-01

    The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle.

  12. Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

    PubMed Central

    Mahata, Bidesh; Zhang, Xiuwei; Kolodziejczyk, Aleksandra A.; Proserpio, Valentina; Haim-Vilmovsky, Liora; Taylor, Angela E.; Hebenstreit, Daniel; Dingler, Felix A.; Moignard, Victoria; Göttgens, Berthold; Arlt, Wiebke; McKenzie, Andrew N.J.; Teichmann, Sarah A.

    2014-01-01

    Summary T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis. PMID:24813893

  13. In situ microscopy analysis reveals local innate immune response developed around Brucella infected cells in resistant and susceptible mice.

    PubMed

    Copin, Richard; Vitry, Marie-Alice; Hanot Mambres, Delphine; Machelart, Arnaud; De Trez, Carl; Vanderwinden, Jean-Marie; Magez, Stefan; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

    2012-01-01

    Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.

  14. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

    PubMed

    Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

    2015-02-01

    A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

  15. Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq.

    PubMed

    Mitchell, Hugh D; Markillie, Lye Meng; Chrisler, William B; Gaffrey, Matthew J; Hu, Dehong; Szymanski, Craig J; Xie, Yumei; Melby, Eric S; Dohnalkova, Alice; Taylor, Ronald C; Grate, Eva K; Cooley, Scott K; McDermott, Jason E; Heredia-Langner, Alejandro; Orr, Galya

    2016-11-22

    The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells "overloaded" while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. Here, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.

  16. Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

    DOE PAGES

    Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; ...

    2016-10-27

    The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantummore » dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.« less

  17. Cells respond to distinct nanoparticle properties with multiple strategies as revealed by single-cell RNA-Seq

    SciTech Connect

    Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; Gaffrey, Matthew J.; Hu, Dehong; Szymanski, Craig J.; Xie, Yumei; Melby, Eric S.; Dohnalkova, Alice; Taylor, Ronald C.; Grate, Eva K.; Cooley, Scott K.; McDermott, Jason E.; Heredia-Langner, Alejandro; Orr, Galya

    2016-10-27

    The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. In this paper, we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with up-regulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly down-regulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong up-regulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis, and organelle activities. In contrast, strategies unique to carboxylated QDs showed up-regulation of DNA repair and RNA activities and decreased regulation of cell division, coupled in some cases with up-regulation of stress responses and ATP-related functions. Finally, together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified proactive defenses or repairs of the NP insults.

  18. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells.

    PubMed

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-09-14

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation.

  19. Measurement of Gene Regulation in Individual Cells Reveals Rapid Switching Between Promoter States

    PubMed Central

    Sepúlveda, Leonardo A.; Xu, Heng; Zhang, Jing; Wang, Mengyu; Golding, Ido

    2016-01-01

    In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, PRM. We simultaneously measured the concentration of the lambda repressor CI and the number of mRNAs from PRM in individual E. coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime, and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits. PMID:26965629

  20. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy.

    PubMed

    Cardoso Dos Santos, Marcelina; Déturche, Régis; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-09-20

    We propose an improved version of variable-angle total internal reflection fluorescence microscopy (vaTIRFM) adapted to modern TIRF setup. This technique involves the recording of a stack of TIRF images, by gradually increasing the incident angle of the light beam on the sample. A comprehensive theory was developed to extract the membrane/substrate separation distance from fluorescently labeled cell membranes. A straightforward image processing was then established to compute the topography of cells with a nanometric axial resolution, typically 10-20 nm. To highlight the new opportunities offered by vaTIRFM to quantify adhesion process of motile cells, adhesion of MDA-MB-231 cancer cells on glass substrate coated with fibronectin was examined.

  1. Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing

    PubMed Central

    Howell, Kristen; Chien, Chun; Bell, Rebecca; Laudier, Damien; Tufa, Sara F.; Keene, Douglas R.; Andarawis-Puri, Nelly; Huang, Alice H.

    2017-01-01

    To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a ‘neo-tendon’ that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing. PMID:28332620

  2. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells

    PubMed Central

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-01-01

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation. PMID:27624869

  3. Mitotic History Reveals Distinct Stem Cell Populations and Their Contributions to Hematopoiesis.

    PubMed

    Säwén, Petter; Lang, Stefan; Mandal, Pankaj; Rossi, Derrick J; Soneji, Shamit; Bryder, David

    2016-03-29

    Homeostasis of short-lived blood cells is dependent on rapid proliferation of immature precursors. Using a conditional histone 2B-mCherry-labeling mouse model, we characterize hematopoietic stem cell (HSC) and progenitor proliferation dynamics in steady state and following several types of induced stress. HSC proliferation following HSC transplantation into lethally irradiated mice is fundamentally different not only from native hematopoiesis but also from other stress contexts. Whereas transplantation promoted sustained, long-term proliferation of HSCs, both cytokine-induced mobilization and acute depletion of selected blood cell lineages elicited very limited recruitment of HSCs to the proliferative pool. By coupling mCherry-based analysis of proliferation history with multiplex gene expression analyses on single cells, we have found that HSCs can be stratified into four distinct subtypes. These subtypes have distinct molecular signatures and differ significantly in their reconstitution potentials, showcasing the power of tracking proliferation history when resolving functional heterogeneity of HSCs.

  4. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  5. Live-cell and super-resolution imaging reveal that the distribution of wall-associated protein A is correlated with the cell chain integrity of Streptococcus mutans.

    PubMed

    Li, Y; Liu, Z; Zhang, Y; Su, Q P; Xue, B; Shao, S; Zhu, Y; Xu, X; Wei, S; Sun, Y

    2015-10-01

    Streptococcus mutans is a primary pathogen responsible for dental caries. It has an outstanding ability to form biofilm, which is vital for virulence. Previous studies have shown that knockout of Wall-associated protein A (WapA) affects cell chain and biofilm formation of S. mutans. As a surface protein, the distribution of WapA remains unknown, but it is important to understand the mechanism underlying the function of WapA. This study applied the fluorescence protein mCherry as a reporter gene to characterize the dynamic distribution of WapA in S. mutans via time-lapse and super-resolution fluorescence imaging. The results revealed interesting subcellular distribution patterns of WapA in single, dividing and long chains of S. mutans cells. It appears at the middle of the cell and moves to the poles as the cell grows and divides. In a cell chain, after each round of cell division, such dynamic relocation results in WapA distribution at the previous cell division sites, resulting in a pattern where WapA is located at the boundary of two adjacent cell pairs. This WapA distribution pattern corresponds to the breaking segmentation of wapA deletion cell chains. The dynamic relocation of WapA through the cell cycle increases our understanding of the mechanism of WapA in maintaining cell chain integrity and biofilm formation.

  6. In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells.

    PubMed

    Blackwood, John K; Williamson, Stuart C; Greaves, Laura C; Wilson, Laura; Rigas, Anastasia C; Sandher, Raveen; Pickard, Robert S; Robson, Craig N; Turnbull, Douglass M; Taylor, Robert W; Heer, Rakesh

    2011-10-01

    Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.

  7. Abrupt and dynamic changes in gene expression revealed by live cell arrays.

    PubMed

    Walling, Maureen A; Shi, Hua; Shepard, Jason R E

    2012-03-20

    A description of the noise associated with gene expression is presented, based on a simplified form of the combined multistep processes of transcription and translation. These processes are influenced by numerous factors, including the accessibility of promoter regions to the transcriptional machinery, the kinetics of assembly of the transcription complexes, and the synthesis and degradation of both mRNA and proteins, among others. Ultimately, stochasticity in cellular processes results in variation in protein levels. Here we constructed a rationally designed RNA-based transcriptional activator to reduce these variables and provide a cleaner, more detailed portrayal of cellular noise. Functioning at a level comparable to natural transcription activation, this activator is isolated to a lacZ reporter gene in yeast cells to quantitatively describe the efficiency of the combined processes of transcription and translation. By employing single-cell array techniques to monitor individual cells simultaneously and in real time, a statistical approach to investigate noise inherent in gene expression is possible. Live cell arrays enabled cell populations to be characterized temporally at the individual cell level. The array platform allowed for a relative measure of protein production in real time and could characterize protein bursts with variable size and random timing, such that bursts occurred in a temporally indiscriminate fashion. The inherent variability and randomness of these processes is characterized, with almost half (47%) of cells experiencing bursting behavior at least once over the course of the experiment. We demonstrate that cells identified on the upper periphery of activity exhibit behaviors that are substantially different from the majority of the population, and such variable activities within a population will provide a more accurate characterization of the population.

  8. A Zebrafish Live Imaging Model Reveals Differential Responses of Microglia Toward Glioblastoma Cells In Vivo

    PubMed Central

    Hamilton, Lloyd; Astell, Katy R.; Velikova, Gergana

    2016-01-01

    Abstract Glioblastoma multiforme is the most common and deadliest form of brain cancer. Glioblastomas are infiltrated by a high number of microglia, which promote tumor growth and surrounding tissue invasion. However, it is unclear how microglia and glioma cells physically interact and if there are differences, depending on glioma cell type. Hence, we have developed a novel live imaging assay to study microglia–glioma interactions in vivo in the zebrafish brain. We transplanted well-established human glioblastoma cell lines, U87 and U251, into transgenic zebrafish lines with labelled macrophages/microglia. Our confocal live imaging results show distinct interactions between microglia and U87, as well as U251 glioblastoma cells that differ in number and nature. Importantly these interactions do not appear to be antitumoral as zebrafish microglia do not engulf and phagocytose the human glioblastoma cells. Finally, xenotransplants into the irf8−/− zebrafish mutant that lacks microglia, as well as pharmacological inhibition of the CSF-1 receptor (CSF-1R) on microglia, confirm a prominent role for zebrafish microglia in promoting human glioblastoma cell growth. This new model will be an important tool for drug screening and the development of future immunotherapeutics targeting microglia within glioma. PMID:27779463

  9. An Atlas of Network Topologies Reveals Design Principles for Caenorhabditis elegans Vulval Precursor Cell Fate Patterning.

    PubMed

    Ping, Xianfeng; Tang, Chao

    2015-01-01

    The vulval precursor cell (VPC) fate patterning in Caenorhabditis elegans is a classic model experimental system for cell fate determination and patterning in development. Despite its apparent simplicity (six neighboring cells arranged in one dimension) and many experimental and computational efforts, the patterning strategy and mechanism remain controversial due to incomplete knowledge of the complex biology. Here, we carry out a comprehensive computational analysis and obtain a reservoir of all possible network topologies that are capable of VPC fate patterning under the simulation of various biological environments and regulatory rules. We identify three patterning strategies: sequential induction, morphogen gradient and lateral antagonism, depending on the features of the signal secreted from the anchor cell. The strategy of lateral antagonism, which has not been reported in previous studies of VPC patterning, employs a mutual inhibition of the 2° cell fate in neighboring cells. Robust topologies are built upon minimal topologies with basic patterning strategies and have more flexible and redundant implementations of modular functions. By simulated mutation, we find that all three strategies can reproduce experimental error patterns of mutants. We show that the topology derived by mapping currently known biochemical pathways to our model matches one of our identified functional topologies. Furthermore, our robustness analysis predicts a possible missing link related to the lateral antagonism strategy. Overall, we provide a theoretical atlas of all possible functional networks in varying environments, which may guide novel discoveries of the biological interactions in vulval development of Caenorhabditis elegans and related species.

  10. Local alignment vectors reveal cancer cell-induced ECM fiber remodeling dynamics.

    PubMed

    Lee, Byoungkoo; Konen, Jessica; Wilkinson, Scott; Marcus, Adam I; Jiang, Yi

    2017-01-03

    Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics.

  11. Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle.

    PubMed

    Pack, Chan Gi; Song, Mi Ryoung; Tae, Eunju Lee; Hiroshima, Michio; Byun, Kyung Hee; Kim, Jun Sung; Sako, Yasushi

    2012-11-10

    For quantitative analysis of nanoparticle diffusions and submicro-environments in living cells, use of newly synthesized silica-based fluorescent nanoparticle (Si-FNP) as a standard nanoprobe is successfully demonstrated. The appropriate characteristics of a standard probe were fully analyzed in vitro by single molecule detection, transmission electron microscopy, and dynamic light scattering. Using fluorescence correlation analysis in single living cells, we quantitatively compared the diffusional properties of the standard Si-FNP with a diameter of 50 nm, peptide coated Si-FNP, streptavidin coated Qdot, and GFP molecule which have different sizes and surface properties. The result demonstrates that the standard Si-FNP without coat is minimally trapped in the vesicles in the process of cellular endocytosis. Interestingly, a large proportion of Si-FNP introduced into the cells by electroporation diffuses freely in the cells during a cell cycle suggesting free diffusing NPs are hardly trapped in the vesicles. The simple but highly sensitive method will provide insight into strategies to understanding the hydrodynamic process of nanoparticle delivery into living cells as well as the cellular microenvironment in the view of submicro-size.

  12. Comprehensive analysis of cellular galectin-3 reveals no consistent oncogenic function in pancreatic cancer cells.

    PubMed

    Hann, Alexander; Gruner, Anja; Chen, Ying; Gress, Thomas M; Buchholz, Malte

    2011-01-01

    Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded.

  13. Comprehensive profiling reveals mechanisms of SOX2-mediated cell fate specification in human ESCs and NPCs

    PubMed Central

    Zhou, Chenlin; Yang, Xiaoqin; Sun, Yiyang; Yu, Hongyao; Zhang, Yong; Jin, Ying

    2016-01-01

    SOX2 is a key regulator of multiple types of stem cells, especially embryonic stem cells (ESCs) and neural progenitor cells (NPCs). Understanding the mechanism underlying the function of SOX2 is of great importance for realizing the full potential of ESCs and NPCs. Here, through genome-wide comparative studies, we show that SOX2 executes its distinct functions in human ESCs (hESCs) and hESC-derived NPCs (hNPCs) through cell type- and stage-dependent transcription programs. Importantly, SOX2 suppresses non-neural lineages in hESCs and regulates neurogenesis from hNPCs by inhibiting canonical Wnt signaling. In hESCs, SOX2 achieves such inhibition by direct transcriptional regulation of important Wnt signaling modulators, WLS and SFRP2. Moreover, SOX2 ensures pluripotent epigenetic landscapes via interacting with histone variant H2A.Z and recruiting polycomb repressor complex 2 to poise developmental genes in hESCs. Together, our results advance our understanding of the mechanism by which cell type-specific transcription factors control lineage-specific gene expression programs and specify cell fate. PMID:26809499

  14. Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B‐cell responses

    PubMed Central

    Veninga, Henrike; Borg, Ellen G. F.; Vreeman, Kyle; Taylor, Philip R.; Kalay, Hakan; van Kooyk, Yvette; Kraal, Georg; Martinez‐Pomares, Luisa

    2015-01-01

    Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169+ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood‐borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169+ macrophages in mice resulted in strong, isotype‐switched, high‐affinity Ab production and the preferential induction and long‐term persistence of Ag‐specific GC B cells and follicular Th cells. In agreement with these observations, CD169+ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B‐cell responses. Our results identify CD169+ macrophages as promoters of high‐affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses. PMID:25487358

  15. Hygroscopic swelling and shrinkage of latewood cell wall micropillars reveal ultrastructural anisotropy.

    PubMed

    Rafsanjani, Ahmad; Stiefel, Michael; Jefimovs, Konstantins; Mokso, Rajmund; Derome, Dominique; Carmeliet, Jan

    2014-06-06

    We document the hygroscopic swelling and shrinkage of the central and the thickest secondary cell wall layer of wood (named S2) in response to changes in environmental humidity using synchrotron radiation-based phase contrast X-ray tomographic nanoscopy. The S2 layer is a natural fibre-reinforced nano-composite polymer and is strongly reactive to water. Using focused ion beam, micropillars with a cross section of few micrometres are fabricated from the S2 layer of the latewood cell walls of Norway spruce softwood. The thin neighbouring cell wall layers are removed to prevent hindering or restraining of moisture-induced deformation during swelling or shrinkage. The proposed experiment intended to get further insights into the microscopic origin of the anisotropic hygro-expansion of wood. It is found that the swelling/shrinkage strains are highly anisotropic in the transverse plane of the cell wall, larger in the normal than in the direction parallel to the cell wall's thickness. This ultrastructural anisotropy may be due to the concentric lamellation of the cellulose microfibrils as the role of the cellulose microfibril angle in the transverse swelling anisotropy is negligible. The volumetric swelling of the cell wall material is found to be substantially larger than the one of wood tissues within the growth ring and wood samples made of several growth rings. The hierarchical configuration in wood optimally increases its dimensional stability in response to a humid environment with higher scales of complexity.

  16. Hygroscopic swelling and shrinkage of latewood cell wall micropillars reveal ultrastructural anisotropy

    PubMed Central

    Rafsanjani, Ahmad; Stiefel, Michael; Jefimovs, Konstantins; Mokso, Rajmund; Derome, Dominique; Carmeliet, Jan

    2014-01-01

    We document the hygroscopic swelling and shrinkage of the central and the thickest secondary cell wall layer of wood (named S2) in response to changes in environmental humidity using synchrotron radiation-based phase contrast X-ray tomographic nanoscopy. The S2 layer is a natural fibre-reinforced nano-composite polymer and is strongly reactive to water. Using focused ion beam, micropillars with a cross section of few micrometres are fabricated from the S2 layer of the latewood cell walls of Norway spruce softwood. The thin neighbouring cell wall layers are removed to prevent hindering or restraining of moisture-induced deformation during swelling or shrinkage. The proposed experiment intended to get further insights into the microscopic origin of the anisotropic hygro-expansion of wood. It is found that the swelling/shrinkage strains are highly anisotropic in the transverse plane of the cell wall, larger in the normal than in the direction parallel to the cell wall's thickness. This ultrastructural anisotropy may be due to the concentric lamellation of the cellulose microfibrils as the role of the cellulose microfibril angle in the transverse swelling anisotropy