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Sample records for 1-positive cells reveals

  1. Two populations of Thy1-positive mesenchymal cells regulate in vitro maturation of hepatic progenitor cells.

    PubMed

    Kamo, Naoko; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Machimoto, Takafumi; Ishii, Takamichi; Fujita, Naoya; Tsuruo, Takashi; Yamashita, Jun K; Kubo, Hajime; Ikai, Iwao

    2007-02-01

    We previously reported that the in vitro maturation of CD49f(+)Thy1(-)CD45(-) (CD49f positive) fetal hepatic progenitor cells (HPCs) is supported by Thy1-positive mesenchymal cells derived from the fetal liver. These mesenchymal cell preparations contain two populations, one of a cuboidal shape and the other spindle shaped in morphology. In this study, we determined that the mucin-type transmembrane glycoprotein gp38 could distinguish cuboidal cells from spindle cells by immunocytochemistry. RT-PCR analysis revealed differences between isolated CD49f(+/-)Thy1(+)gp38(+)CD45(-) (gp38 positive) cells and CD49f(+/-)Thy1(+)gp38(-)CD45(-) (gp38 negative) cells, whereas both cells expressed mesenchymal cell markers. The coculture with gp38-positive cells promoted the maturation of CD49f-positive HPCs, which was estimated by positivity for periodic acid-Schiff (PAS) staining, whereas the coculture with gp38-negative cells maintained CD49f-positive HPCs negative for PAS staining. The expression of mature hepatocyte markers, such as tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and glucose-6-phosphatase, were upregulated on HPCs by coculture with gp38-positive cells. Furthermore, transmission electron microscopy revealed the acquisition of mature hepatocyte features by HPCs cocultured with gp38-positive cells. This effect on maturation of HPCs was inhibited by the addition of conditioned medium derived from gp38-negative cells. By contrast, the upregulation of bromodeoxyuridine incorporation by HPCs demonstrated the proliferative effect of coculture with gp38-negative cells. In conclusion, these results suggest that in vitro maturation of HPCs promoted by gp38-positive cells may be opposed by an inhibitory effect of gp38-negative cells, which likely maintain the immature, proliferative state of HPCs. PMID:16990447

  2. 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. PMID:26840832

  3. 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

  4. A Case of Lymphomatoid Papulosis Occurred Simultaneously with Ki-1-Positive Anaplastic Large Cell Lymphoma

    PubMed Central

    Lee, Nam-Su; Cha, Sang-Woo; Hong, Su-Jin; Shin, Won-Yong; Lee, Gyu-Taeg; Jeon, Jin-Woo; Won, Jong-Ho; Baick, Seung-Ho; Hong, Dae-Sik; Park, Hee-Sook

    1997-01-01

    Lymphomatoid papulosis (LyP) is a chronic self-healing skin eruption that is clinically benign but histologically mimics a malignant lymphoma. However, lymphomatoid papulosis with anaplastic large cell lymphoma responds poorly to medical treatments, including chemotherapies. We experienced a 60-year-old male patient with lymphomatoid papulosis occurred simultaneously with relapsed Ki-1-positive anaplastic large cell lymphoma who was treated with salvage chemotherapy but, unfortunately, failed to be rescued. We report it with a review of the literature. PMID:9159045

  5. Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro.

    PubMed

    Yamamoto, Kimiko; Sokabe, Takaaki; Watabe, Tetsuro; Miyazono, Kohei; Yamashita, Jun K; Obi, Syotaro; Ohura, Norihiko; Matsushita, Akiko; Kamiya, Akira; Ando, Joji

    2005-04-01

    Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1(+)) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G(2)-M phases of the cell cycle than Flk-1(+) ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle alpha-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1(+) ES cells into the endothelial cell lineage. The shear stressed Flk-1(+) ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1(+) ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1(+) ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1(+) ES cells. PMID:15576436

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

    PubMed Central

    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

  7. 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. PMID:18094147

  8. PINK1 positively regulates HDAC3 to suppress dopaminergic neuronal cell death.

    PubMed

    Choi, Hyo-Kyoung; Choi, Youngsok; Kang, HeeBum; Lim, Eun-Jin; Park, Soo-Yeon; Lee, Hyun-Seob; Park, Ji-Min; Moon, Jisook; Kim, Yoon-Jung; Choi, Insup; Joe, Eun-Hye; Choi, Kyung-Chul; Yoon, Ho-Geun

    2015-02-15

    Deciphering the molecular basis of neuronal cell death is a central issue in the etiology of neurodegenerative diseases, such as Parkinson's and Alzheimer's. Dysregulation of p53 levels has been implicated in neuronal apoptosis. The role of histone deacetylase 3 (HDAC3) in suppressing p53-dependent apoptosis has been recently emphasized; however, the molecular basis of modulation of p53 function by HDAC3 remains unclear. Here we show that PTEN-induced putative kinase 1 (PINK1), which is linked to autosomal recessive early-onset familial Parkinson's disease, phosphorylates HDAC3 at Ser-424 to enhance its HDAC activity in a neural cell-specific manner. PINK1 prevents H2O2-induced C-terminal cleavage of HDAC3 via phosphorylation of HDAC3 at Ser-424, which is reversed by protein phosphatase 4c. PINK1-mediated phosphorylation of HDAC3 enhances its direct association with p53 and causes subsequent hypoacetylation of p53. Genetic deletion of PINK1 partly impaired the suppressive role of HDAC3 in regulating p53 acetylation and transcriptional activity. However, depletion of HDAC3 fully abolished the PINK1-mediated p53 inhibitory loop. Finally, ectopic expression of phosphomometic-HDAC3(S424E) substantially overcomes the defective action of PINK1 against oxidative stress in dopaminergic neuronal cells. Together, our results uncovered a mechanism by which PINK1-HDAC3 network mediates p53 inhibitory loop in response to oxidative stress-induced damage. PMID:25305081

  9. Inhibition of cell proliferation by ciprofibrate in glutathione S-transferase P1-1-positive rat hepatic hyperplastic nodules.

    PubMed

    Chen, Z Y; Liu, Y F; He, C Y; White, C C; Eaton, D L

    1994-05-15

    Previous studies have demonstrated that short-term treatment with a peroxisome proliferator (PP) decreased the size and number of genotoxic carcinogen-induced hepatic hyperplastic lesions identified by gamma-glutamyl transpeptidase (GGT) or glutathione S-transferase P1-1 (rGSTP1-1) staining. However, longer-term PP treatment of animals bearing similar hepatic hyperplastic lesions produced an increase in both the size and number of liver tumors. To characterize the hepatic hyperplastic lesions which are inhibited or promoted by PP, a unique double labeling technique was developed to determine the relative rate of cell division (e.g., DNA synthesis) in rGSTP1-1-positive nodules before and after ciprofibrate (Cip) treatment. rGSTP1-1-positive nodules were induced with the Solt-Farber resistance protocol (diethylnitrosamine-2-acetylaminofluorene partial hepatectomy). Eleven weeks after diethylnitrosamine initiation, 3 groups of rats were maintained on a control chow diet or switched to a powdered chow diet containing 0.025% Cip or 0.05% phenobarbital (PB) for the last 8 days of the experiment. A minipump implanted in the abdominal cavity released [methyl-3H]thymidine continuously for 72 h and was then removed prior to CIp or PB treatment. A second minipump was then implanted which released bromodeoxyuridine to the abdominal cavity 5 days after the start of Cip or PB administration and lasted for 72 h until the termination of the experiment. Both the [methyl-3H]thymidine and bromodeoxyuridine labeling indices (LIs) were determined in the same group of cells within individual rGSTP1-1-positive nodules in the right posterior lobes of livers. PB treatment increased both the average number of persistent GGT-positive nodules and the ratio of persistent GGT-positive to rGSTP1-1-positive nodules/cm2. In contrast, Cip treatment greatly decreased the average number and area of persistent GGT-positive nodules, as well as the ratio between persistent GGT-positive and rGSTP1-1

  10. IL-13-producing BLT1-positive CD8 cells are increased in asthma and are associated with airway obstruction

    PubMed Central

    Dakhama, Azzeddine; Collins, Maureen L.; Ohnishi, Hiroshi; Goleva, Elena; Leung, Donald Y. M.; Alam, Rafeul; Sutherland, E. Rand; Martin, Richard J.; Gelfand, Erwin W.

    2013-01-01

    Background The role of CD8 T lymphocytes in the pathogenesis of asthma is not well understood. We investigated whether a subset of IL-13-producing BLT1-positive CD8 T lymphocytes is present in asthmatic airways and is associated with impaired lung function. Methods Bronchoalveolar lavage (BAL) cells were obtained from asthmatic (n=39) and healthy control (n=28) subjects. Cells were stimulated with phorbol ester and ionomycin in the presence of brefeldin A and stained for CD8, BLT1 and intracellular IL-13. The frequency of IL-13-producing BLT1-positive CD8 T lymphocytes was compared between the two groups and related to lung function, serum IgE levels and reticular basement membrane (RBM) thickness. Results A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 was detected in the airways of all asthmatic subjects. The frequency of this subset among recovered lymphocytes was significantly higher in the airways of asthmatic subjects compared to controls (mean ± SEM: 16.2 ± 1.4 vs. 5.3 ± 0.5, respectively, p < 0.001), and correlated positively with serum IgE levels and RBM thickness. More importantly, the frequency of CD8 T lymphocytes co-expressing BLT1 and IL-13 was inversely related to FEV1 and FEF[25-75] percent predicted values (p<0.001). Conclusions A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 is present in the airways of asthmatics. The accumulation of these cells is associated with airway obstruction, suggesting that they may play a significant pathogenic role in bronchial asthma. PMID:23573812

  11. Derivation and Isolation of NKX2.1-Positive Basal Forebrain Progenitors from Human Embryonic Stem Cells

    PubMed Central

    Germain, Noélle D.; Banda, Erin C.; Becker, Sandy; Naegele, Janice R.

    2013-01-01

    Gamma aminobutyric acid (GABA)-expressing interneurons are the major inhibitory cells of the cerebral cortex and hippocampus. These interneurons originate in the medial ganglionic eminence (MGE) and lateral ganglionic eminence of the ventral forebrain during embryonic development and show reduced survival and function in a variety of neurological disorders, including temporal lobe epilepsy. We and others have proposed that embryonic stem cell (ESC)–derived ventral forebrain progenitors might provide a source of new GABAergic interneurons for cell-based therapies. While human ESCs (hESCs) are readily differentiated in vitro into dorsal telencephalic neural progenitors, standard protocols for generating ventral subtypes of telencephalic progenitors are less effective. We now report efficient derivation of GABAergic progenitors using an established hESC reporter line that expresses green fluorescent protein (GFP) under the control of an endogenous NKX2.1 promoter. GABAergic progenitors were derived from this hESC line by a modified monolayer neural differentiation protocol. Consistent with sonic hedgehog (SHH)-dependent specification of NKX2.1-positive progenitors in the embryonic MGE, we show a dose-dependent increase in the generation of NKX2.1:GFP-positive progenitors after SHH treatment in vitro. Characterization of NKX2.1:GFP-positive cells confirms their identity as MGE-like neural progenitors, based on gene expression profiles and their ability to differentiate into GABAergic interneurons. We are also able to generate highly enriched populations of NKX2.1:GFP-positive progenitors, including cells with telencephalic identity, by fluorescence-activated cell sorting. These hESC-derived ventral forebrain progenitors are suitable candidates for cell-based therapies that aim at replacing dysfunctional or damaged cortical or hippocampal GABAergic interneurons. PMID:23351095

  12. Metformin increases antitumor activity of MEK inhibitors through GLI1 downregulation in LKB1 positive human NSCLC cancer cells

    PubMed Central

    Della Corte, Carminia Maria; Ciaramella, Vincenza; Mauro, Concetta Di; Castellone, Maria Domenica; Papaccio, Federica; Fasano, Morena; Sasso, Ferdinando Carlo; Martinelli, Erika; Troiani, Teresa; De Vita, Ferdinando; Orditura, Michele; Bianco, Roberto; Ciardiello, Fortunato; Morgillo, Floriana

    2016-01-01

    Purpose Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1-wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. Experimental design Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene. Results The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters. Conclusions Metformin potentiates the antitumor activity of MEK-Is in human LKB1-wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9. PMID:26673006

  13. HTLV-1 positive and negative T cells cloned from infected individuals display telomerase and telomere genes deregulation that predominate in activated but untransformed CD4+ T cells.

    PubMed

    Zane, Linda; Sibon, David; Capraro, Valérie; Galia, Perrine; Karam, Maroun; Delfau-Larue, Marie-Hélène; Gilson, Eric; Gessain, Antoine; Gout, Olivier; Hermine, Olivier; Mortreux, Franck; Wattel, Eric

    2012-08-15

    Untransformed HTLV-1 positive CD4(+) cells from infected individuals are selected for expressing tax and displaying morphological features consistent with telomere dysfunctions. We show that in resting HTLV-1 positive CD4(+) cells cloned from patients, hTERT expression parallels tax expression and cell cycling. Upon activation, these cells dramatically augment tax expression, whereas their increase in telomerase activity is about 20 times lower than that of their uninfected counterpart. Activated HTLV-1 positive CD4(+) but not uninfected CD4(+) or CD8(+) clones also repress the transcription of TRF1, TPP1, TANK1, POT1, DNA-PKc and Ku80. Both infected and uninfected lymphocytes from infected individuals shared common telomere gene deregulations toward a pattern consistent with premature senescence. ATLL cells displayed the highest telomerase activity (TA) whereas recovered a telomere gene transcriptome close to that of normal CD4(+) cells. In conclusion HTLV-1-dependent telomere modulations seem involved in clonal expansion, immunosuppression, tumor initiation and progression. PMID:21717459

  14. miR-146b-5p within BCR-ABL1-Positive Microvesicles Promotes Leukemic Transformation of Hematopoietic Cells.

    PubMed

    Zhang, Hong-Mei; Li, Qing; Zhu, Xiaojian; Liu, Wei; Hu, Hui; Liu, Teng; Cheng, Fanjun; You, Yong; Zhong, Zhaodong; Zou, Ping; Li, Qiubai; Chen, Zhichao; Guo, An-Yuan

    2016-05-15

    Evidence is accumulating that extracellular microvesicles (MV) facilitate progression and relapse in cancer. Using a model in which MVs derived from K562 chronic myelogenous leukemia (CML) cells transform normal hematopoietic transplants into leukemia-like cells, we defined the underlying mechanisms of this process through gene-expression studies and network analyses of transcription factors (TF) and miRNAs. We found that antitumor miRNAs were increased and several defense pathways were initiated during the early phases of oncogenic transformation. Later, oncomiRs and genes involved in cell cycle, DNA repair, and energy metabolism pathways were upregulated. Regulatory network analyses revealed that a number of TFs and miRNAs were responsible for the pathway dysregulation and the oncogenic transformation. In particular, we found that miR-146b-5p, which was highly expressed in MVs, coordinated the regulation of cancer-related genes to promote cell-transforming processes. Notably, treatment of recipient cells with MV derived from K562 cells expressing mimics of miR-146b-5p revealed that it accelerated the transformation process in large part by silencing the tumor-suppressor NUMB High levels of miR-146b-5p also enhanced reactive oxygen species levels and genome instability of recipient cells. Taken together, our finding showed how upregulation of oncogenic miRNAs in MVs promote hematopoetic cells to a leukemic state, as well as a demonstration for TF and miRNA coregulatory analysis in exploring the dysregulation of cancers and discovering key factors. Cancer Res; 76(10); 2901-11. ©2016 AACR. PMID:27013199

  15. A novel molecular variant of the chimeric NPM-ALK transcript in Ki-1 positive large cell lymphoma

    SciTech Connect

    Ladanyi, M.

    1994-09-01

    The breakpoints of the t(2;5)(p23;q35), reported to be present in 30-40% of Ki-1 positive large cell lymphoma (Ki-1 LCL), have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 cases with the translocation. We performed NPM-ALK RT-PCR on 16 cases of Ki-1 LCL, of which 13 had informative cytogenetics. Amplifiable template was confirmed in all samples by RT-PCR using {beta}-actin primers. Ten cases showed the expected 177 base pair (bp) RT-PCR product indicative of the translocation, including all 6 cases with cytogenetic evidence of the t(2;5) and 3 cases without 5q35 abnormalities. One additional case, which had uninformative cytogenetics, showed only a novel 144 bp RT-PCR product. Sequencing of this PCR product showed an in-frame junction of NPM to ALK, 20 bp distal to the usual NPM junction site and 53 bp distal to usual ALK junction site. The predicted chimeric protein is thus shorter by 11 amino acids, but the putative ALK catalytic domain remains intact. This case was a histologically typical anaplastic Ki-1 LCL which showed a clonal rearrangement of the T-cell receptor {beta} gene. The functional significance of this molecular variant and its relation to the exon structure of both genes require further study. The overall incidence of the translocation in this series, about 70%, is higher than suspected from cytogenetic analysis alone.

  16. The absence of aldehyde dehydrogenase 1 A1-positive cells in benign mammary stroma is associated with risk factors for breast cancer

    PubMed Central

    Isfoss, Björn Logi; Holmqvist, Bo; Jernström, Helena; Alm, Per; Olsson, Håkan

    2016-01-01

    In this study, aldehyde dehydrogenase 1 (ALDH1)-expressing cells in stroma of histologically normal breast tissue from premenopausal women were investigated in situ regarding cellular morphology, cell distribution, and relation to the additional stem cell markers, CD44 (+) and CD24 (−). These results were correlated with hormonal and genetic risk factors for breast cancer. Triple immunofluorescence labeling was performed on tissues from premenopausal women with a family history of breast cancer, and breast reduction specimens from premenopausal women with no family history of breast cancer were used as a control group. The majority of ALDH1-immunoreactive cells in stroma were spindle-shaped or polygonal, and such cells that were CD44− and CD24− were absent in the breast stroma of a significantly larger number of nulliparous than parous women. A less common morphological type of ALDH1-positive cells in stroma was round or oval in shape, and such cells that were CD44+ and CD24− were absent in a significant number of women with a family history of breast cancer. The CD44+/CD24− immunophenotype is consistent with stem cells, and the round/oval morphology suggests mesenchymal cells. This study demonstrates that there are two morphologically distinct types of ALDH1-positive cells in histologically benign mammary stroma, and the absence of these cells is correlated with clinical risk factors for breast cancer in premenopausal women. PMID:27313475

  17. The absence of aldehyde dehydrogenase 1 A1-positive cells in benign mammary stroma is associated with risk factors for breast cancer.

    PubMed

    Isfoss, Björn Logi; Holmqvist, Bo; Jernström, Helena; Alm, Per; Olsson, Håkan

    2016-01-01

    In this study, aldehyde dehydrogenase 1 (ALDH1)-expressing cells in stroma of histologically normal breast tissue from premenopausal women were investigated in situ regarding cellular morphology, cell distribution, and relation to the additional stem cell markers, CD44 (+) and CD24 (-). These results were correlated with hormonal and genetic risk factors for breast cancer. Triple immunofluorescence labeling was performed on tissues from premenopausal women with a family history of breast cancer, and breast reduction specimens from premenopausal women with no family history of breast cancer were used as a control group. The majority of ALDH1-immunoreactive cells in stroma were spindle-shaped or polygonal, and such cells that were CD44(-) and CD24(-) were absent in the breast stroma of a significantly larger number of nulliparous than parous women. A less common morphological type of ALDH1-positive cells in stroma was round or oval in shape, and such cells that were CD44(+) and CD24(-) were absent in a significant number of women with a family history of breast cancer. The CD44(+)/CD24(-) immunophenotype is consistent with stem cells, and the round/oval morphology suggests mesenchymal cells. This study demonstrates that there are two morphologically distinct types of ALDH1-positive cells in histologically benign mammary stroma, and the absence of these cells is correlated with clinical risk factors for breast cancer in premenopausal women. PMID:27313475

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

    PubMed Central

    Debald, Manuel; Rostamzadeh, Babak; Thiesler, Thore; Schröder, Lars; Barchet, Winfried; Abramian, Alina; Kaiser, Christina; Kristiansen, Glen; Kuhn, Walther; Kübler, Kirsten

    2015-01-01

    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. PMID:25999351

  19. 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. PMID:25999351

  20. GABABR-Dependent Long-Term Depression at Hippocampal Synapses between CB1-Positive Interneurons and CA1 Pyramidal Cells

    PubMed Central

    Jappy, Dave; Valiullina, Fliza; Draguhn, Andreas; Rozov, Andrei

    2016-01-01

    Activity induced long lasting modifications of synaptic efficacy have been extensively studied in excitatory synapses, however, long term plasticity is also a property of inhibitory synapses. Inhibitory neurons in the hippocampal CA1 region can be subdivided according to the compartment they target on the pyramidal cell. Some interneurons preferentially innervate the perisomatic area and axon hillock of the pyramidal cells while others preferentially target dendritic branches and spines. Another characteristic feature allowing functional classification of interneurons is cell type specific expression of different neurochemical markers and receptors. In the hippocampal CA1 region, nearly 90% of the interneurons expressing cannabinoid type 1 receptors (CB1R) also express cholecystokinin (CCK). Therefore, the functional presence of CB1 receptors can be used for identification of the inhibitory input from CCK positive (CCK+) interneurons to CA1 pyramidal cells. The goal of this study was to explore the nature of long term plasticity at the synapses between interneurons expressing CB1Rs (putative CCK+) and pyramidal neurons in the CA1 region of the hippocampus in vitro. We found that theta burst stimulation triggered robust long-term depression (LTD) at this synapse. The locus of LTD induction was postsynaptic and required activation of GABAB receptors. We also showed that LTD at this synaptic connection involves GABABR-dependent suppression of adenylyl cyclase and consequent reduction of PKA activity. In this respect, CB1+ to pyramidal cell synapses differ from the majority of the other hippocampal inhibitory connections where theta burst stimulation results in long-term potentiation. PMID:26858602

  1. GABABR-Dependent Long-Term Depression at Hippocampal Synapses between CB1-Positive Interneurons and CA1 Pyramidal Cells.

    PubMed

    Jappy, Dave; Valiullina, Fliza; Draguhn, Andreas; Rozov, Andrei

    2016-01-01

    Activity induced long lasting modifications of synaptic efficacy have been extensively studied in excitatory synapses, however, long term plasticity is also a property of inhibitory synapses. Inhibitory neurons in the hippocampal CA1 region can be subdivided according to the compartment they target on the pyramidal cell. Some interneurons preferentially innervate the perisomatic area and axon hillock of the pyramidal cells while others preferentially target dendritic branches and spines. Another characteristic feature allowing functional classification of interneurons is cell type specific expression of different neurochemical markers and receptors. In the hippocampal CA1 region, nearly 90% of the interneurons expressing cannabinoid type 1 receptors (CB1R) also express cholecystokinin (CCK). Therefore, the functional presence of CB1 receptors can be used for identification of the inhibitory input from CCK positive (CCK+) interneurons to CA1 pyramidal cells. The goal of this study was to explore the nature of long term plasticity at the synapses between interneurons expressing CB1Rs (putative CCK+) and pyramidal neurons in the CA1 region of the hippocampus in vitro. We found that theta burst stimulation triggered robust long-term depression (LTD) at this synapse. The locus of LTD induction was postsynaptic and required activation of GABAB receptors. We also showed that LTD at this synaptic connection involves GABABR-dependent suppression of adenylyl cyclase and consequent reduction of PKA activity. In this respect, CB1+ to pyramidal cell synapses differ from the majority of the other hippocampal inhibitory connections where theta burst stimulation results in long-term potentiation. PMID:26858602

  2. FOXA1 positively regulates gene expression by changing gene methylation status in human breast cancer MCF-7 cells

    PubMed Central

    Zheng, Lu; Qian, Bo; Tian, Duo; Tang, Tong; Wan, Shengyun; Wang, Lei; Zhu, Lixin; Geng, Xiaoping

    2015-01-01

    Objective: DNA methylation is an important epigenetic modification with tumor suppressor gene silencing in cancer. The mechanisms underlying DNA methylation patterns are still poorly understood. This study aims to evaluate the potential value of FOXA1 for controlling gene CpG island methylation in breast cancer. Methods: FOXA1 was down-regulated by transfection with siRNA and up-regulated by transfection with plasmid in MCF-7 cell lines. The DNA methylation and mRNA levels were examined by qMSP and qRT-PCR. The cell proliferation and apoptosis was detected by MTT and Flow cytometry. Results: Suppression of FOXA1 enhanced the methylation status of DAPK, MGMT, RASSF1A, p53, and depressed mRNA levels of these tumor suppressor genes, whereas over-expression of FOXA1 showed the opposite effects. DNMT1, DNMT3A and DNMT3B mRNA were up-regulated by siRNA knock-down of FOXA1. At the same time, FOXA1 suppression promoted cell growth and inhibited apoptosis. Conclusions: FOXA1 may be associated with methylation of the tumor suppressor genes promoter through changing DNMTs expression. FOXA1 could be a potential demethylation target for prevention and treatment of breast cancer. PMID:25755696

  3. Primary Ureteral Thyroid Transcription Factor 1-Positive Small Cell Neuroendocrine Carcinoma: Case Report and Review of the Literature.

    PubMed

    Acosta, Andrés Martin; Hamedani, Farid Saei; Meeks, Joshua James; Wu, Shoujin

    2015-09-01

    Upper urinary tract neoplasms comprise only 5% of urinary malignancies overall, the overwhelming majority showing an urothelial histology. Nonurothelial lesions represent 1.9% of upper tract neoplasms, and small cell carcinomas (SmCCs) only account for a minute fraction of them, with few cases reported in the literature to date. In the genitourinary tract, these lesions are most frequently found in the urinary bladder, where they tend to show a mixed histology with a characteristic small cell neuroendocrine component that is positive for chromogranin A, synaptophysin, and CD56 immunostains. Urinary SmCCs are almost invariably aggressive, with a propensity to infiltrate surrounding tissues and vascular structures. Unfortunately, clinical stage at presentation is often advanced, with early dissemination to lymph nodes or distant organs in a significant proportion of patients. For the most part, prognosis is dismal and can be predicted by the pathologic stage at diagnosis. Recently, new chemotherapeutic regimens have shown some potential benefit for the treatment of urinary SmCC, albeit not achieving curative results. Here we present the case of a 71-year-old male patient with a history of treated bladder urothelial carcinoma that presented with subacute flank pain and was diagnosed with a primary pure SmCC of the ureter. Besides the usual neuroendocrine immunomarkers, this tumor showed intense positivity for TTF-1 (thyroid transcription factor-1), a phenomenon that has been described for extrapulmonary SmCC of other locations. PMID:26162396

  4. Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells.

    PubMed

    Roy, Ananya; Coum, Antoine; Marinescu, Voichita D; Põlajeva, Jelena; Smits, Anja; Nelander, Sven; Uhrbom, Lene; Westermark, Bengt; Forsberg-Nilsson, Karin; Pontén, Fredrik; Tchougounova, Elena

    2015-09-15

    Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials. PMID:26164207

  5. 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. PMID:27066979

  6. Effect of Nerium oleander (N.O.) Leaves Extract on Serum hepcidin, Total Iron, and Infiltration of ED1 Positive Cells in Albino Rat

    PubMed Central

    Abbasi, Muddasir Hassan; Fatima, Sana; Naz, Naila; Malik, Ihtzaz A.

    2013-01-01

    To gain insight into the hepatohistological alterations in noninjured rat liver, Nerium oleander (N.O.) leaves extract was injected intramuscularly to induce an acute phase reaction (APR). Histopathological changes were studied after 3, 12, and 24 h time course of sterile muscle abscess. Tissue integrity and any infiltration of inflammatory cells in the liver were investigated by Hematoxylin and Eosin and ED1 peroxidase stainings. The administration of N.O. leaves extract (10 mL/kg) in H & E stained sections showed a general vacuolization of cytoplasm resulting loss of polarity with prominent nucleoli after 3 h of induction. At 12 h, eccentric nuclei were also observed in the sections. Marked infiltration of leucocytes with predominate macrophages was also found after 24 h as seen by ED1 positive staining. In the present study, a possible relationship between serum hepcidin and total iron level was also investigated in vivo. An early increase of hepcidin and total iron level (3 h) with a maximum at 12 h (P < 0.01; P < 0.001) was observed. These changes indicate that sterile muscle abscess may induce APR resulting in hepatic damage which is evident with the recruitment of inflammatory cells into the organ. PMID:24069586

  7. Siglec-1-positive plasmacytoid dendritic cells (pDCs) in human peripheral blood: A semi-mature and myeloid-like subset imbalanced during protective and autoimmune responses.

    PubMed

    Wilhelm, Theresa R; Taddeo, Adriano; Winter, Oliver; Schulz, Axel Ronald; Mälzer, Julia-Nora; Domingo, Cristina; Biesen, Robert; Alexander, Tobias; Thiel, Andreas; Radbruch, Andreas; Hiepe, Falk; Gerl, Velia

    2016-02-01

    Plasmacytoid dendritic cells (pDCs) play a central role in the pathogenesis of systemic lupus erythematosus (SLE) as IFN-α producers and promoters of T-cell activation or tolerance. Here, we demonstrated by flow-cytometry and confocal microscopy that Siglec-1, a molecule involved in the regulation of adaptive immunoresponses, is expressed in a subset of semi-mature, myeloid-like pDCs in human blood. These pDCs express lower BDCA-2 and CD123 and higher HLA-DR and CD11c than Siglec-1-negative pDCs and do not produce IFN-α via TLR7/TLR9 engagement. In vitro, Siglec-1 expression was induced in Siglec-1-negative pDCs by influenza virus. Proportions of Siglec-1-positive/Siglec-1-negative pDCs were higher in SLE than in healthy controls and correlated with disease activity. Healthy donors immunized with yellow fever vaccine YFV-17D displayed different kinetics of the two pDC subsets during protective immune response. PDCs can be subdivided into two subsets according to Siglec-1 expression. These subsets may play specific roles in (auto)immune responses. PMID:26674280

  8. A High-avidity WT1-reactive T-Cell Receptor Mediates Recognition of Peptide and Processed Antigen but not Naturally Occurring WT1-positive Tumor Cells.

    PubMed

    Jaigirdar, Adnan; Rosenberg, Steven A; Parkhurst, Maria

    2016-04-01

    Wilms tumor gene 1 (WT1) is an attractive target antigen for cancer immunotherapy because it is overexpressed in many hematologic malignancies and solid tumors but has limited, low-level expression in normal adult tissues. Multiple HLA class I and class II restricted epitopes have been identified in WT1, and multiple investigators are pursuing the treatment of cancer patients with WT1-based vaccines and adoptively transferred WT1-reactive T cells. Here we isolated an HLA-A*0201-restricted WT1-reactive T-cell receptor (TCR) by stimulating peripheral blood lymphocytes of healthy donors with the peptide WT1:126-134 in vitro. This TCR mediated peptide recognition down to a concentration of ∼0.1 ng/mL when pulsed onto T2 cells as well as recognition of HLA-A*0201 target cells transfected with full-length WT1 cDNA. However, it did not mediate consistent recognition of many HLA-A*0201 tumor cell lines or freshly isolated leukemia cells that endogeneously expressed WT1. We dissected this pattern of recognition further and observed that WT1:126-134 was more efficiently processed by immunoproteasomes compared with standard proteasomes. However, pretreatment of WT1 tumor cell lines with interferon gamma did not appreciably enhance recognition by our TCR. In addition, we highly overexpressed WT1 in several leukemia cell lines by electroporation with full-length WT1 cDNA. Some of these lines were still not recognized by our TCR suggesting possible antigen processing defects in some leukemias. These results suggest WT1:126-134 may not be a suitable target for T-cell based tumor immunotherapies. PMID:26938944

  9. 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

  10. Obg-like ATPase 1 regulates global protein serine/threonine phosphorylation in cancer cells by suppressing the GSK3β-inhibitor 2-PP1 positive feedback loop

    PubMed Central

    Xu, Dong; Song, Renduo; Wang, Guohui; Jeyabal, Prince V.S.; Weiskoff, Amanda M.; Ding, Kefeng; Shi, Zheng-Zheng

    2016-01-01

    OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif. PMID:26655089

  11. TTF-1-positive oncocytic sellar tumor with follicle formation/ependymal differentiation: non-adenomatous tumor capable of two different interpretations as a pituicytoma or a spindle cell oncocytoma.

    PubMed

    Yoshimoto, Toyoki; Takahashi-Fujigasaki, Junko; Inoshita, Naoko; Fukuhara, Noriaki; Nishioka, Hiroshi; Yamada, Shozo

    2015-07-01

    We describe herein the unique case of a 70-year-old male with a TTF-1-positive non-adenomatous sellar tumor that has unusual morphological and immunohistochemical features. MRI examination detected a 2-cm sellar mass that was enhanced heterogeneously. By histology, the tumor was composed of epithelioid and oncocytic cells arranged in a trabecular pattern with occasional luminal structures. The lesion was diffusely immunopositive for thyroid transcription factor-1 (TTF-1) and vimentin but negative for S100 protein and GFAP. Immunoreactivity for epithelial membrane antigen, low molecular weight cytokeratin (CAM 5.2), and neuronal markers was also observed in the tumor cells. By electron microscopy, the tumor cells were filled with abundant mitochondria and extended microvillous projections into small extracellular and intracellular lumens. TTF-1 is considered to be an excellent marker of pituicytes, specialized glia of the neurohypophysis. This case can be regarded as a variant of pituicytoma, showing both ependymal differentiation and oncocytic changes. However, the immunoprofile was not completely consistent with a pituicyte lineage; the epithelial features suggested a possibility of folliculostellate cell origin. TTF-1-positive sellar neoplasms might therefore have variable morphological and immunohistochemical profiles. For suitable classification of TTF-1 positive sellar neoplasms, their histological features should be carefully re-evaluated. PMID:25893822

  12. Conditional Mutagenesis of Gata6 in SF1-Positive Cells Causes Gonadal-Like Differentiation in the Adrenal Cortex of Mice

    PubMed Central

    Pihlajoki, Marjut; Gretzinger, Elisabeth; Cochran, Rebecca; Kyrönlahti, Antti; Schrade, Anja; Hiller, Theresa; Sullivan, Laura; Shoykhet, Michael; Schoeller, Erica L.; Brooks, Michael D.; Heikinheimo, Markku

    2013-01-01

    Transcription factor GATA6 is expressed in the fetal and adult adrenal cortex and has been implicated in steroidogenesis. To characterize the role of transcription factor GATA6 in adrenocortical development and function, we generated mice in which Gata6 was conditionally deleted using Cre-LoxP recombination with Sf1-cre. The adrenal glands of adult Gata6 conditional knockout (cKO) mice were small and had a thin cortex. Cytomegalic changes were evident in fetal and adult cKO adrenal glands, and chromaffin cells were ectopically located at the periphery of the glands. Corticosterone secretion in response to exogenous ACTH was blunted in cKO mice. Spindle-shaped cells expressing Gata4, a marker of gonadal stroma, accumulated in the adrenal subcapsule of Gata6 cKO mice. RNA analysis demonstrated the concomitant upregulation of other gonadal-like markers, including Amhr2, in the cKO adrenal glands, suggesting that GATA6 inhibits the spontaneous differentiation of adrenocortical stem/progenitor cells into gonadal-like cells. Lhcgr and Cyp17 were overexpressed in the adrenal glands of gonadectomized cKO vs control mice, implying that GATA6 also limits sex steroidogenic cell differentiation in response to the hormonal changes that accompany gonadectomy. Nulliparous female and orchiectomized male Gata6 cKO mice lacked an adrenal X-zone. Microarray hybridization identified Pik3c2g as a novel X-zone marker that is downregulated in the adrenal glands of these mice. Our findings offer genetic proof that GATA6 regulates the differentiation of steroidogenic progenitors into adrenocortical cells. PMID:23471215

  13. 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. PMID:26456260

  14. 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. PMID:27117143

  15. 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

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

    PubMed

    Kippner, Linda E; Kim, Jinhee; Gibson, Greg; Kemp, Melissa L

    2014-01-01

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

  17. Micropatterning of cells reveals chiral morphogenesis

    PubMed Central

    2013-01-01

    Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development. PMID:23672821

  18. Cell-size maintenance: universal strategy revealed.

    PubMed

    Jun, Suckjoon; Taheri-Araghi, Sattar

    2015-01-01

    How cells maintain a stable size has fascinated scientists since the beginning of modern biology, but has remained largely mysterious. Recently, however, the ability to analyze single bacteria in real time has provided new, important quantitative insights into this long-standing question in cell biology. PMID:25497321

  19. Relief of diabetes by duodenal-jejunal bypass sleeve implantation in the high-fat diet and streptozotocin-induced diabetic rat model is associated with an increase in GLP-1 levels and the number of GLP-1-positive cells

    PubMed Central

    SHUANG, JINQUAN; ZHANG, YING; MA, LIMEI; TAN, XUEMING; HUANG, JING; WANG, XIANG; XIONG, GUANYIN; JIANG, ZHONGHUA; ZHANG, XIUHUA; DU, SHIQING; GU, YONGSONG; SHI, XIANGYANG; FAN, ZHINING

    2015-01-01

    A recently invented duodenal-jejunal bypass sleeve (DJBS) implanted in the duodenum and proximal jejunum has exhibited good glycemic control in diabetes mellitus. However, the specific mechanism by which DJBS placement induces the remission of diabetes is not well known. Previous studies have indicated that changes in the pattern of gut hormone secretion may play a role. The aim of the present study was to explore the role of intestinal L cells and the production of glucagon-like peptide-1 (GLP-1) by these cells in DJBS implantation-induced glycemic control in diabetic rats. A DJBS was placed in the proximal small intestine of rats with diabetes induced by a high-fat diet and low-dose streptozotocin (STZ), and the effects of the DJBS on the remission of diabetes and the GLP-1 levels of plasma and intestinal tissues were investigated 12 weeks after DJBS placement. The number of intestinal GLP-1 positive cells was also counted. When the DJBS had been in place for 12 weeks, the plasma glucose level of the DJBS-implanted rats decreased significantly from 23.33±1.56 mmol/l prior to surgery to 7.70±0.84 mmol/l and the diabetes mellitus was relieved completely; however, diabetic control rats and diabetic rats subjected to sham surgery did not show any improvement. Parallel with the remission of diabetes, the plasma and distal ileum GLP-1 levels of rats in the DJBS implantation group were also higher than those of rats in the diabetic control and sham surgery groups. The number of GLP-1-positive cells in the distal ileum was also higher in the DJBS implantation group than in the diabetic control and sham surgery groups (31.0±2.6 vs. 23.5±4.4 vs. 23.0±3.2 respectively; P<0.01). DJBS implantation effectively led to the remission of diabetes in rats with diabetes induced by a high-fat diet and low-dose STZ when implanted for 12 weeks. The remission of diabetes may be associated with the increase in the number of L cells and elevation of GLP-1 levels induced by DJBS

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

    PubMed

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

    2016-02-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. We constructed a cellular taxonomy of one cortical region, primary visual cortex, in adult mice on the basis of single-cell RNA sequencing. We identified 49 transcriptomic cell types, including 23 GABAergic, 19 glutamatergic and 7 non-neuronal types. We also analyzed cell type-specific mRNA processing and characterized genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we found that some of our transcriptomic cell types displayed specific and differential electrophysiological and axon projection properties, thereby confirming that the single-cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  1. Laparoscopic sterilization in HIV-1-positive women.

    PubMed

    Intaraprasert, S; Taneepanichskul, S; Chaturachinda, K

    1996-11-01

    Laparoscopic sterilizations in HIV-1-positive women were performed. Patients, who were HIV-1-positive, underwent voluntary laparoscopic sterilization. The mean age of patients was 27.5 +/- 3.8 years. Most were of low socioeconomic status. The mean duration of the operation was 14.4 +/- 5.4 min. No accidental injury to the surgical team was recorded, and no complications occurred among the patients. It was concluded that laparoscopic sterilization in HIV-positive patients was safe with low risk of HIV transmission to the surgical team. PMID:8934065

  2. 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'. PMID:26083756

  3. Automated live cell imaging systems reveal dynamic cell behavior.

    PubMed

    Chirieleison, Steven M; Bissell, Taylor A; Scelfo, Christopher C; Anderson, Jordan E; Li, Yong; Koebler, Doug J; Deasy, Bridget M

    2011-07-01

    Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity. PMID:21692197

  4. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

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

  7. Melanoma Cell Colony Expansion Parameters Revealed by Approximate Bayesian Computation

    PubMed Central

    Vo, Brenda N.; Drovandi, Christopher C.; Pettitt, Anthony N.; Pettet, Graeme J.

    2015-01-01

    In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies. This can improve our understanding of cancer formation and progression. Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data. To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation. Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2–12%. The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not. The posterior mean values of D and q are in the ranges 226–268 µm2h−1, 311–351 µm2h−1 and 0.23–0.39, 0.32–0.61 for the experimental periods of 0–24 h and 24–48 h, respectively. Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not. The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ. PMID:26642072

  8. Melanoma Cell Colony Expansion Parameters Revealed by Approximate Bayesian Computation.

    PubMed

    Vo, Brenda N; Drovandi, Christopher C; Pettitt, Anthony N; Pettet, Graeme J

    2015-12-01

    In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies. This can improve our understanding of cancer formation and progression. Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data. To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation. Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2-12%. The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not. The posterior mean values of D and q are in the ranges 226-268 µm2h-1, 311-351 µm2h-1 and 0.23-0.39, 0.32-0.61 for the experimental periods of 0-24 h and 24-48 h, respectively. Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not. The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ. PMID:26642072

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Macaulay, Iain C.; Svensson, Valentine; Labalette, Charlotte; Ferreira, Lauren; Hamey, Fiona; Voet, Thierry; Teichmann, Sarah A.; Cvejic, Ana

    2016-01-01

    Summary The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment. PMID:26804912

  11. 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

  12. Mpath maps multi-branching single-cell trajectories revealing progenitor cell progression during development

    PubMed Central

    Chen, Jinmiao; Schlitzer, Andreas; Chakarov, Svetoslav; Ginhoux, Florent; Poidinger, Michael

    2016-01-01

    Single-cell RNA-sequencing offers unprecedented resolution of the continuum of state transition during cell differentiation and development. However, tools for constructing multi-branching cell lineages from single-cell data are limited. Here we present Mpath, an algorithm that derives multi-branching developmental trajectories using neighborhood-based cell state transitions. Applied to mouse conventional dendritic cell (cDC) progenitors, Mpath constructs multi-branching trajectories spanning from macrophage/DC progenitors through common DC progenitor to pre-dendritic cells (preDC). The Mpath-generated trajectories detect a branching event at the preDC stage revealing preDC subsets that are exclusively committed to cDC1 or cDC2 lineages. Reordering cells along cDC development reveals sequential waves of gene regulation and temporal coupling between cell cycle and cDC differentiation. Applied to human myoblasts, Mpath recapitulates the time course of myoblast differentiation and isolates a branch of non-muscle cells involved in the differentiation. Our study shows that Mpath is a useful tool for constructing cell lineages from single-cell data. PMID:27356503

  13. Mpath maps multi-branching single-cell trajectories revealing progenitor cell progression during development.

    PubMed

    Chen, Jinmiao; Schlitzer, Andreas; Chakarov, Svetoslav; Ginhoux, Florent; Poidinger, Michael

    2016-01-01

    Single-cell RNA-sequencing offers unprecedented resolution of the continuum of state transition during cell differentiation and development. However, tools for constructing multi-branching cell lineages from single-cell data are limited. Here we present Mpath, an algorithm that derives multi-branching developmental trajectories using neighborhood-based cell state transitions. Applied to mouse conventional dendritic cell (cDC) progenitors, Mpath constructs multi-branching trajectories spanning from macrophage/DC progenitors through common DC progenitor to pre-dendritic cells (preDC). The Mpath-generated trajectories detect a branching event at the preDC stage revealing preDC subsets that are exclusively committed to cDC1 or cDC2 lineages. Reordering cells along cDC development reveals sequential waves of gene regulation and temporal coupling between cell cycle and cDC differentiation. Applied to human myoblasts, Mpath recapitulates the time course of myoblast differentiation and isolates a branch of non-muscle cells involved in the differentiation. Our study shows that Mpath is a useful tool for constructing cell lineages from single-cell data. PMID:27356503

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

    PubMed

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

    2016-07-01

    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

  15. 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

  16. 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. PMID:26000486

  17. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  18. Cell-by-Cell Dissection of Gene Expression and Chromosomal Interactions Reveals Consequences of Nuclear Reorganization

    PubMed Central

    2005-01-01

    The functional consequences of long-range nuclear reorganization were studied in a cell-by-cell analysis of gene expression and long-range chromosomal interactions in the Drosophila eye and eye imaginal disk. Position-effect variegation was used to stochastically perturb gene expression and probe nuclear reorganization. Variegating genes on rearrangements of Chromosomes X, 2, and 3 were probed for long-range interactions with heterochromatin. Studies were conducted only in tissues known to express the variegating genes. Nuclear structure was revealed by fluorescence in situ hybridization with probes to the variegating gene and heterochromatin. Gene expression was determined alternately by immunofluorescence against specific proteins and by eye pigment autofluorescence. This allowed cell-by-cell comparisons of nuclear architecture between cells in which the variegating gene was either expressed or silenced. Very strong correlations between heterochromatic association and silencing were found. Expressing cells showed a broad distribution of distances between variegating genes and their own centromeric heterochromatin, while silenced cells showed a very tight distribution centered around very short distances, consistent with interaction between the silenced genes and heterochromatin. Spatial and temporal analysis of interactions with heterochromatin indicated that variegating genes primarily associate with heterochromatin in cells that have exited the cell cycle. Differentiation was not a requirement for association, and no differences in association were observed between cell types. Thus, long-range interactions between distal chromosome regions and their own heterochromatin have functional consequences for the organism. PMID:15737020

  19. 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

  20. 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.

  1. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance

    PubMed Central

    Steinert, Elizabeth M.; Schenkel, Jason M.; Fraser, Kathryn A.; Beura, Lalit K.; Manlove, Luke S.; Igyártó, Botond Z.; Southern, Peter J.; Masopust, David

    2015-01-01

    Summary Memory CD8 T cells protect against intracellular pathogens by scanning host cell surfaces, thus infection detection rates depend on memory cell number and distribution. Population analyses rely on isolation from whole organs and interpretation is predicated on presumptions of near complete cell recovery. Paradigmatically, memory is parsed into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns, but in practice identified by phenotypic markers. Because isolation methods ultimately inform models of memory T cell differentiation, protection, and vaccine translation, we tested their validity via parabiosis and quantitative immunofluorescence microscopy of a mouse memory CD8 T cell population. We report three major findings: lymphocyte isolation fails to recover most cells and biases against certain subsets, residents greatly outnumber recirculating cells within nonlymphoid tissues, and memory subset homing to inflammation does not conform to previously hypothesized migration patterns. These results indicate that most host cells are surveyed for reinfection by segregated residents rather than by recirculating cells that migrate throughout the blood and body. PMID:25957682

  2. 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

  3. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

    PubMed

    Trapnell, Cole; Cacchiarelli, Davide; Grimsby, Jonna; Pokharel, Prapti; Li, Shuqiang; Morse, Michael; Lennon, Niall J; Livak, Kenneth J; Mikkelsen, Tarjei S; Rinn, John L

    2014-04-01

    Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation. PMID:24658644

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

    PubMed Central

    2010-01-01

    Background The term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs). Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature. PMID:20465783

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2014-11-30

    Neuroblastoma (NB), an embryonal tumour of the sympathetic nervous system, is thought to originate from undifferentiated neural crest cells and is known to exhibit extremely heterogeneous biological and clinical behaviors. Occurring in very young children, the median age at diagnosis is 17 months and it accounts for 10% of all pediatric cancer mortalities. The standard treatment regimen for patients with high-risk NB includes induction and surgery followed by isotretinoin or Accutane (13-cis retinoic acid) treatment, which is shown to induce terminal differentiation of NB cells. However, molecular regulators that maintain an undifferentiated phenotype in NB cells are still poorly understood. PMID:25483101

  7. A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

    PubMed Central

    Vizeacoumar, Franco J; Arnold, Roland; Vizeacoumar, Frederick S; Chandrashekhar, Megha; Buzina, Alla; Young, Jordan T F; Kwan, Julian H M; Sayad, Azin; Mero, Patricia; Lawo, Steffen; Tanaka, Hiromasa; Brown, Kevin R; Baryshnikova, Anastasia; Mak, Anthony B; Fedyshyn, Yaroslav; Wang, Yadong; Brito, Glauber C; Kasimer, Dahlia; Makhnevych, Taras; Ketela, Troy; Datti, Alessandro; Babu, Mohan; Emili, Andrew; Pelletier, Laurence; Wrana, Jeff; Wainberg, Zev; Kim, Philip M; Rottapel, Robert; O'Brien, Catherine A; Andrews, Brenda; Boone, Charles; Moffat, Jason

    2013-01-01

    Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model. PMID:24104479

  8. Single-cell dynamics reveals sustained growth during diauxic shifts.

    PubMed

    Boulineau, Sarah; Tostevin, Filipe; Kiviet, Daniel J; ten Wolde, Pieter Rein; Nghe, Philippe; Tans, Sander J

    2013-01-01

    Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival. PMID:23637881

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

    PubMed Central

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

    2015-01-01

    Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo. PMID:25941375

  10. [Ph1 positive acute lymphoblastic leukemia with DIC after operation of colon and lung cancer].

    PubMed

    Yashige, H; Fujii, H

    1989-07-01

    We reported a rare case of triple cancers with acute lymphoblastic leukemia (ALL) associated with disseminated intravascular coagulopathy (DIC) after the operations of colon cancer and primary lung cancer. A 78-year-old Japanese male, who had been operated upon for colon cancer (adenocarcinoma) on March 1981, metastatic brain tumor (adenocarcinoma) on December 1986, and primary lung cancer (squamous cell carcinoma) on February 1987, was admitted to our hospital because of severe general malaise on December 6 1987. On admission, he had mild hepatosplenomegaly and hemorrhage diathesis such as purpura. Serum LDH increased to 2,515 mU/ml. The white blood cell count was 6,210/microliters with 53% leukemia cells, and the platelet count was 12,000/microliters. A bone marrow was infiltrated with 96.0% leukemia cells. The leukemia cells stained positively for PAS and negatively for peroxidase. Immunological examination of leukemia cells showed that HLA-DR, TdT, B1 and J5 were positive and cytoplasmic Igmu and surface Ig were negative, indicating common ALL. The coagulation studies revealed that the activated partial thromboplastin time was prolonged to 42.0 seconds, FDP increased to 79.9 micrograms/ml, and antithrombin-III decreased to 62%. Chromosome analysis showed a 48, XY, +2, +21q-, t(9;22) karyotype. He was diagnosed as having Ph1 positive ALL associated with DIC. He was treated with vindesine, prednisolone, L-asparaginase, and adriamycin and complete remission (CR) was achieved after two months. But on August 1988, 8 months after CR, ALL and brain tumor relapsed and he died of pneumonia on September 19, 1988. PMID:2810793

  11. Phase Resetting Reveals Network Dynamics Underlying a Bacterial Cell Cycle

    PubMed Central

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R.; Scherer, Norbert F.

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS). PMID:23209388

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

    PubMed Central

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

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation is presented. SHG-based three-dimensional reconstructions of Chlamydomonas cells containing starch granules are shown. PMID:23009858

  13. 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. PMID:22266279

  14. 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

  15. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    PubMed Central

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

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

    PubMed

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

    2015-10-22

    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. PMID:26375009

  17. 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.

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

    DOE PAGESBeta

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

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

    PubMed Central

    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. PMID:26790980

  20. 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.

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

    PubMed Central

    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

    Abstract 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. PMID:19845817

  2. Pure red-cell aplasia "epidemic"--mystery completely revealed?

    PubMed

    Locatelli, Francesco; Del Vecchio, Lucia; Pozzoni, Pietro

    2007-06-01

    Starting in 1998, the number of pure red-cell aplasia (PRCA) cases in patients treated with recombinant human erythropoietin (rHuEPO) increased dramatically. Most cases were observed in patients treated with epoetin alfa produced outside the United States. The peak was observed in 2002; since then, the PRCA incidence has declined. Many factors are likely to have contributed to this up-surge. The molecular structure of the various epoetins and patient characteristics do not seem to play a major role. The route of administration holds some importance, because most PRCA patients received rHuEPO subcutaneously. The peak of PRCA cases overlapped with the removal of human serum albumin from the Eprex formulation (Janssen-Pharmaceutica NV, Beerse, Belgium), for which polysorbate 80 and glycine were substituted. Polysorbate 80 may have increased the immunogenicity of Eprex by eliciting the formation of epoetin-containing micelles or by interacting with leachates released by the uncoated rubber stoppers of prefilled syringes. Compared with the previous formulation, the polysorbate 80 formulation has lower stability, making it more susceptible to stress conditions such as insufficient attention to the cold chain. This situation could facilitate protein denaturation or aggregate formation. Uncoated rubber stoppers were replaced with coated stoppers, and the cold chain was reinforced; the Eprex formulation has remained unchanged. Even though the incidence of PRCA returned to very low levels, discriminating the cause-effect relationship of a single action is difficult, given that all occurred with a similar chronology, and that PRCA develops after a relatively long exposure period. Careful observation of future trends of new PRCA cases is thus mandatory. PMID:17556324

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

    PubMed

    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

    2015-07-01

    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

  4. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

    NASA Astrophysics Data System (ADS)

    Dvir, Liron; Nissim, Ronen; Alvarez-Elizondo, Martha B.; Weihs, Daphne

    2015-04-01

    Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

  5. 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. PMID:24919153

  6. 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.

  7. 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. PMID:19074636

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

    PubMed Central

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

    2009-01-01

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

  9. Whole Genome Expression Analysis Reveals Differential Effects of TiO2 Nanotubes on Vascular Cells

    PubMed Central

    Peng, Lily; Barczak, Andrea J.; Barbeau, Rebecca A.; Xiao, Yuanyuan; LaTempa, Thomas J.; Grimes, Craig A.; Desai, Tejal A.

    2010-01-01

    The response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to TiO2 nanotube arrays is studied through gene expression analysis. Microarrays revealed that nanotubes enhanced EC proliferation and motility, decreased VSMC proliferation, and decreased expression of molecules involved in inflammation and coagulation in both cell types. Networks generated from significantly affected genes suggest that cells may be sensing nanotopographical cues via pathways previously implicated in sensing shear stress. PMID:20030358

  10. Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells

    PubMed Central

    Ju, Z.; Kapoor, M.; Newton, K; Cheon, K.; Ramaswamy, A.; Lotan, R.; Strong, L. C.; Koo, J. S.

    2006-01-01

    To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a > 1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium. PMID:16049682

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

    PubMed

    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

  12. 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

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

    PubMed

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

    2016-03-01

    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

  14. 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. PMID:27425622

  15. Mechanisms of Cell Cycle Control Revealed by a Systematic and Quantitative Overexpression Screen in S. cerevisiae

    PubMed Central

    Niu, Wei; Li, Zhihua; Zhan, Wenjing; Iyer, Vishwanath R.; Marcotte, Edward M.

    2008-01-01

    Regulation of cell cycle progression is fundamental to cell health and reproduction, and failures in this process are associated with many human diseases. Much of our knowledge of cell cycle regulators derives from loss-of-function studies. To reveal new cell cycle regulatory genes that are difficult to identify in loss-of-function studies, we performed a near-genome-wide flow cytometry assay of yeast gene overexpression-induced cell cycle delay phenotypes. We identified 108 genes whose overexpression significantly delayed the progression of the yeast cell cycle at a specific stage. Many of the genes are newly implicated in cell cycle progression, for example SKO1, RFA1, and YPR015C. The overexpression of RFA1 or YPR015C delayed the cell cycle at G2/M phases by disrupting spindle attachment to chromosomes and activating the DNA damage checkpoint, respectively. In contrast, overexpression of the transcription factor SKO1 arrests cells at G1 phase by activating the pheromone response pathway, revealing new cross-talk between osmotic sensing and mating. More generally, 92%–94% of the genes exhibit distinct phenotypes when overexpressed as compared to their corresponding deletion mutants, supporting the notion that many genes may gain functions upon overexpression. This work thus implicates new genes in cell cycle progression, complements previous screens, and lays the foundation for future experiments to define more precisely roles for these genes in cell cycle progression. PMID:18617996

  16. Mathematical Modeling Reveals That Changes to Local Cell Density Dynamically Modulate Baseline Variations in Cell Growth and Drug Response.

    PubMed

    Greene, James M; Levy, Doron; Herrada, Sylvia P; Gottesman, Michael M; Lavi, Orit

    2016-05-15

    Cell-to-cell variations contribute to drug resistance with consequent therapy failure in cancer. Experimental techniques have been developed to monitor tumor heterogeneity, but estimates of cell-to-cell variation typically fail to account for the expected spatiotemporal variations during the cell growth process. To fully capture the extent of such dynamic variations, we developed a mechanistic mathematical model supported by in vitro experiments with an ovarian cancer cell line. We introduce the notion of dynamic baseline cell-to-cell variation, showing how the emerging spatiotemporal heterogeneity of one cell population can be attributed to differences in local cell density and cell cycle. Manipulation of the geometric arrangement and spatial density of cancer cells revealed that given a fixed global cell density, significant differences in growth, proliferation, and paclitaxel-induced apoptosis rates were observed based solely on cell movement and local conditions. We conclude that any statistical estimate of changes in the level of heterogeneity should be integrated with the dynamics and spatial effects of the baseline system. This approach incorporates experimental and theoretical methods to systematically analyze biologic phenomena and merits consideration as an underlying reference model for cell biology studies that investigate dynamic processes affecting cancer cell behavior. Cancer Res; 76(10); 2882-90. ©2016 AACR. PMID:26933088

  17. 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

  18. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.

    PubMed

    Hauser, Anja E; Junt, Tobias; Mempel, Thorsten R; Sneddon, Michael W; Kleinstein, Steven H; Henrickson, Sarah E; von Andrian, Ulrich H; Shlomchik, Mark J; Haberman, Ann M

    2007-05-01

    Proliferation, mutation, and selection in the germinal center (GC) are thought to occur in distinct microanatomical compartments-the dark zone (DZ) and the light zone (LZ). Thus, affinity maturation has been posited to require frequent trafficking between zones. Here we report the use of multiphoton in vivo microscopy to determine migration patterns of GC B cells. Analysis of time-resolved images revealed unexpected patterns of movement as well as GC B cell morphology. Though frequent movement between the DZ and LZ was anticipated, few cells were observed to cross the interface between the two compartments. Moreover, cell-track trajectories indicated that cell movement in this region is predominantly parallel to the interface, suggesting that B cells circulate within individual LZ and DZ compartments. The results suggest a revision to our views of B cell circulation within GCs and the functional relationship of its two major compartments. PMID:17509908

  19. 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. PMID:27074779

  20. Controlled One-on-One Encounters between Immune Cells and Microbes Reveal Mechanisms of Phagocytosis.

    PubMed

    Heinrich, Volkmar

    2015-08-01

    Among many challenges facing the battle against infectious disease, one quandary stands out. On the one hand, it is often unclear how well animal models and cell lines mimic human immune behavior. On the other hand, many core methods of cell and molecular biology cannot be applied to human subjects. For example, the profound susceptibility of neutropenic patients to infection marks neutrophils (the most abundant white blood cells in humans) as vital immune defenders. Yet because these cells cannot be cultured or genetically manipulated, there are gaps in our understanding of the behavior of human neutrophils. Here, we discuss an alternative, interdisciplinary strategy to dissect fundamental mechanisms of immune-cell interactions with bacteria and fungi. We show how biophysical analyses of single-live-cell/single-target encounters are revealing universal principles of immune-cell phagocytosis, while also dispelling misconceptions about the minimum required mechanistic determinants of this process. PMID:26244729

  1. Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.

    PubMed

    Jakštys, Baltramiejus; Ruzgys, Paulius; Tamošiūnas, Mindaugas; Šatkauskas, Saulius

    2015-10-01

    The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results. PMID:26077843

  2. Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

    PubMed Central

    Welker, Alessandra M.; Jaros, Brian D.; Puduvalli, Vinay K.; Imitola, Jaime; Kaur, Balveen; Beattie, Christine E.

    2016-01-01

    ABSTRACT Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for

  3. Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death

    PubMed Central

    2016-01-01

    Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death. PMID:25965523

  4. 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

    2015-05-01

    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. PMID:25954881

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

    PubMed Central

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

    2015-01-01

    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. PMID:25954881

  6. 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. PMID:27547806

  7. 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.

  8. 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

  9. 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

  10. Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

    PubMed

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

    2009-08-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a

  11. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

  14. 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

  15. Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling

    PubMed Central

    Geoghegan, Vincent; Guo, Ailan; Trudgian, David; Thomas, Benjamin; Acuto, Oreste

    2015-01-01

    The impact of protein arginine methylation on the regulation of immune functions is virtually unknown. Here, we apply a novel method—isomethionine methyl-SILAC—coupled with antibody-mediated arginine-methylated peptide enrichment to identify methylated peptides in human T cells by mass spectrometry. This approach allowed the identification of 2,502 arginine methylation sites from 1,257 tissue-specific and housekeeping proteins. We find that components of T cell antigen receptor signal machinery and several key transcription factors that regulate T cell fate determination are methylated on arginine. Moreover, we demonstrate changes in arginine methylation stoichiometry during cellular stimulation in a subset of proteins critical to T cell differentiation. Our data suggest that protein arginine methyltransferases exert key regulatory roles in T cell activation and differentiation, opening a new field of investigation in T cell biology. PMID:25849564

  16. An efficient method that reveals both the dendrites and the soma mosaics of retinal ganglion cells.

    PubMed

    Zhan, X J; Troy, J B

    1997-03-01

    A method of using neurobiotin to stain both the dendrites and the soma mosaics of retinal ganglion cells in fresh retinae is described. This method is simple to use and efficient in revealing morphological details for a large number of retinal ganglion cells. It has five advantages over currently available staining methods. (1) It stains all ganglion cells in the whole retina or in a selected retinal area, permitting ganglion cell distributions across the retina to be obtained. (2) It reveals cell dendrites in great detail, especially in regions outside the area centralis. The dendritic field mosaics and, therefore the dendritic field coverage factors, of different ganglion cell types across the whole retina can be obtained easily. (3) It works reliably, efficiently, and does not require the expensive set-up or the pains-taking work needed when staining cells through intracellular injection. (4) It works under both in vivo and in vitro settings, permitting the use of retinae from animals sacrificed for other purposes and the use of postmortem human retinae. (5) The end product of the visualization process is optically dark and electron dense, permitting specimens to be examined under both light and electron microscopes. PMID:9128174

  17. 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

  18. Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration

    PubMed Central

    Bennett, Christopher G.; Riemondy, Kent; Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong; Kuersten, Scott; Yi, Rui

    2016-01-01

    The Musashi-2 (Msi2) RNA-binding protein maintains stem cell self-renewal and promotes oncogenesis by enhancing cell proliferation in hematopoietic and gastrointestinal tissues. However, it is unclear how Msi2 recognizes and regulates mRNA targets in vivo and whether Msi2 primarily controls cell growth in all cell types. Here we identified Msi2 targets with HITS-CLIP and revealed that Msi2 primarily recognizes mRNA 3′UTRs at sites enriched in multiple copies of UAG motifs in epithelial progenitor cells. RNA-seq and ribosome profiling demonstrated that Msi2 promotes targeted mRNA decay without affecting translation efficiency. Unexpectedly, the most prominent Msi2 targets identified are key regulators that govern cell motility with a high enrichment in focal adhesion and extracellular matrix-receptor interaction, in addition to regulators of cell growth and survival. Loss of Msi2 stimulates epithelial cell migration, increases the number of focal adhesions and also compromises cell growth. These findings provide new insights into the molecular mechanisms of Msi2's recognition and repression of targets and uncover a key function of Msi2 in restricting epithelial cell migration. PMID:27034466

  19. An optimized isolation of biotinylated cell surface proteins reveals novel players in cancer metastasis

    PubMed Central

    Karhemo, Piia-Riitta; Ravela, Suvi; Laakso, Marko; Ritamo, Ilja; Tatti, Olga; Mäkinen, Selina; Goodison, Steve; Stenman, Ulf-Håkan; Hölttä, Erkki; Hautaniemi, Sampsa; Valmu, Leena; Lehti, Kaisa; Laakkonen, Pirjo

    2012-01-01

    Details of metastasis, the deadliest aspect of cancer, are unclear. Cell surface proteins play central roles in adhesive contacts between the tumor cell and the stroma during metastasis. We optimized a fast, small-scale isolation of biotinylated cell surface proteins to reveal novel metastasis-associated players froman isogenic pair of human MDA-MB-435 cancer cells with opposite metastatic phenotypes. Isolated proteins were trypsin digested and analyzed using LC–MS/MS followed by quantitation with the Progenesis LC–MS software. Sixteen proteins displayed over twofold expression differences between the metastatic and non-metastatic cells. Interestingly, overexpression of most of them (14/16) in the metastatic cells indicates a gain of novel surface protein profile as compared to the non-metastatic one. All five validated, differentially expressed proteins showed higher expression in the metastatic cells in culture, and four of these were further validated in vivo. Moreover, we analyzed the expression of two of the identified proteins, CD109 and ITGA6 in 3-dimensional cultures of six melanoma cell lines. Both proteins marked the surface of cells derived from melanoma metastasis over cells derived from primary melanoma. These unbiased identification and validation of both known and novel metastasis-associated proteins indicate a reliable approach for the identification of differentially expressed surface proteins. PMID:22813880

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

    PubMed Central

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

    2015-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of 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. Stem Cells 2015;33:988–998 PMID:25447755

  1. Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response

    PubMed Central

    Feinerman, Ofer; Jentsch, Garrit; Tkach, Karen E; Coward, Jesse W; Hathorn, Matthew M; Sneddon, Michael W; Emonet, Thierry; Smith, Kendall A; Altan-Bonnet, Grégoire

    2010-01-01

    Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL-2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression. PMID:21119631

  2. Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types.

    PubMed

    Lopez, M J; Upchurch, B H; Rindi, G; Leiter, A B

    1995-01-13

    We have produced transgenic mice expressing fusion genes consisting of 1.6 kilobase pairs of the secretin gene 5' flanking region to direct the expression of human growth hormone (hGH) or simian virus 40 large T antigen to secretin-producing cells. Analysis of different mouse tissues for hGH transcripts revealed expression in each of the major secretin-producing tissues, namely the intestine and endocrine pancrease. Multiple label immunohistochemistry demonstrated that the transgene was correctly directed to secretin cells in the intestinal tract, including a previously unrecognized population of secretin cells in the colon of adult and developing mice. In the small intestine, subpopulations of hGH-containing cells frequently coexpressed substance P, serotonin, and cholecystokinin, whereas in the colon, cells expressing hGH frequently coexpressed glucagon, peptide YY, or neurotensin. Transgenic mice expressing large T antigen in secretin cells developed poorly differentiated neuroendocrine tumors of the small intestine, well differentiated colonic tumors containing glucagon-expressing cells, and insulin-producing tumors in pancreas. These studies indicate that the major cis-regulatory sequences necessary for secretin expression in enteroendocrine cells and fetal islets are localized with 1.6 kilobase pairs of the transcriptional start site. Coexpression of reporter transgenes with several gastrointestinal hormones suggests a potential relationships between secretin cells and other enteroendocrine cell types, as well as pancreatic beta cells. PMID:7822327

  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. Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

    PubMed Central

    Kaplinsky, Joseph; Li, Anthony; Sun, Amy; Coffre, Maryaline; Koralov, Sergei B.; Arnaout, Ramy

    2014-01-01

    Antibody repertoires are known to be shaped by selection for antigen binding. Unexpectedly, we now show that selection also acts on a non–antigen-binding antibody region: the heavy-chain variable (VH)–encoded “elbow” between variable and constant domains. By sequencing 2.8 million recombined heavy-chain genes from immature and mature B-cell subsets in mice, we demonstrate a striking gradient in VH gene use as pre-B cells mature into follicular and then into marginal zone B cells. Cells whose antibodies use VH genes that encode a more flexible elbow are more likely to mature. This effect is distinct from, and exceeds in magnitude, previously described maturation-associated changes in heavy-chain complementarity determining region 3, a key antigen-binding region, which arise from junctional diversity rather than differential VH gene use. Thus, deep sequencing reveals a previously unidentified mode of B-cell selection. PMID:24927543

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2015-11-15

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

  7. 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

  8. Cancer cell(s) cycle sequencing reveals universal mechanisms of apoptosis.

    PubMed

    Marretta, R M Ardito; Ales, F

    2010-12-01

    In this paper, cell cycle in higher eukaryotes and their molecular networks signals both in G1/S and G2/M transitions are replicated in silico. Biochemical kinetics, converted into a set of differential equations, and system control theory are employed to design multi-nested digital layers to simulate protein-to-protein activation and inhibition for cell cycle dynamics in the presence of damaged genomes. Sequencing and controlling the digital process of four micro-scale species networks (p53/Mdm2/DNA damage, p21mRNA/cyclin-CDK complex, CDK/CDC25/weel/SKP2/APC/CKI and apoptosis target genes system) not only allows the comprehension of the mechanisms of these molecule interactions but paves the way for unraveling the participants and their by-products, until now quite unclear, which have the task of carrying out (or not) cell death. Whatever the running simulations (e.g., different species signals, mutant cells and different DNA damage levels), the results of the proposed cell digital multi-layers give reason to believe in the existence of a universal apoptotic mechanism. As a consequence, we identified and selected cell check points, sizers, timers and specific target genes dynamic both for influencing mitotic process and avoiding cancer proliferation as much as for leading the cancer cell(s) to collapse into a steady stable apoptosis phase. PMID:21141676

  9. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

    PubMed Central

    Horowitz, Amir; Strauss-Albee, Dara M.; Leipold, Michael; Kubo, Jessica; Nemat-Gorgani, Neda; Dogan, Ozge C.; Dekker, Cornelia L.; Mackey, Sally; Maecker, Holden; Swan, Gary E.; Davis, Mark M.; Norman, Paul J.; Guethlein, Lisbeth A.; Desai, Manisha; Parham, Peter; Blish, Catherine A.

    2013-01-01

    Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on peripheral blood NK cells from five sets of monozygotic twins and twelve unrelated donors of defined HLA and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6,000-30,000 phenotypic populations within an individual and >100,000 phenotypes in this population. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors, while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation. PMID:24154599

  10. 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

  11. 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. PMID:27200348

  12. 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

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

    PubMed Central

    Papatsenko, Dmitri; Darr, Henia; Kulakovskiy, Ivan V.; Waghray, Avinash; Makeev, Vsevolod J.; MacArthur, Ben D.; Lemischka, Ihor R.

    2015-01-01

    Summary Analyses of gene expression in single mouse embryonic stem cells (mESCs) cultured in serum and LIF revealed the presence of two distinct cell subpopulations with individual gene expression signatures. Comparisons with published data revealed that cells in the first subpopulation are phenotypically similar to cells isolated from the inner cell mass (ICM). In contrast, cells in the second subpopulation appear to be more mature. Pluripotency Gene Regulatory Network (PGRN) reconstruction based on single-cell data and published data suggested antagonistic roles for Oct4 and Nanog in the maintenance of pluripotency states. Integrated analyses of published genomic binding (ChIP) data strongly supported this observation. Certain target genes alternatively regulated by OCT4 and NANOG, such as Sall4 and Zscan10, feed back into the top hierarchical regulator Oct4. Analyses of such incoherent feedforward loops with feedback (iFFL-FB) suggest a dynamic model for the maintenance of mESC pluripotency and self-renewal. PMID:26267829

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

    PubMed

    Kim, Tae-Hee; Saadatpour, Assieh; Guo, Guoji; Saxena, Madhurima; Cavazza, Alessia; Desai, Niyati; Jadhav, Unmesh; Jiang, Lan; Rivera, Miguel N; Orkin, Stuart H; Yuan, Guo-Cheng; Shivdasani, Ramesh A

    2016-08-23

    Lgr5(+) intestinal stem cells (ISCs) drive epithelial self-renewal, and their immediate progeny-intestinal bipotential progenitors-produce absorptive and secretory lineages via lateral inhibition. To define features of early transit from the ISC compartment, we used a microfluidics approach to measure selected stem- and lineage-specific transcripts in single Lgr5(+) cells. We identified two distinct cell populations, one that expresses known ISC markers and a second, abundant population that simultaneously expresses markers of stem and mature absorptive and secretory cells. Single-molecule mRNA in situ hybridization and immunofluorescence verified expression of lineage-restricted genes in a subset of Lgr5(+) cells in vivo. Transcriptional network analysis revealed that one group of Lgr5(+) cells arises from the other and displays characteristics expected of bipotential progenitors, including activation of Notch ligand and cell-cycle-inhibitor genes. These findings define the earliest steps in ISC differentiation and reveal multilineage gene priming as a fundamental property of the process. PMID:27524622

  15. Hematopoietic Signaling Mechanism Revealed from a Stem/Progenitor Cell Cistrome.

    PubMed

    Hewitt, Kyle J; Kim, Duk Hyoung; Devadas, Prithvia; Prathibha, Rajalekshmi; Zuo, Chandler; Sanalkumar, Rajendran; Johnson, Kirby D; Kang, Yoon-A; Kim, Jin-Soo; Dewey, Colin N; Keles, Sunduz; Bresnick, Emery H

    2015-07-01

    Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology. PMID:26073540

  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

    2015-09-01

    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. PMID:26299571

  17. In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay.

    PubMed

    Uchida, Aya; Kishi, Kasane; Aiyama, Yoshimi; Miura, Kento; Takase, Hinako M; Suzuki, Hitomi; Kanai-Azuma, Masami; Iwamori, Tokuko; Kurohmaru, Masamichi; Tsunekawa, Naoki; Kanai, Yoshiakira

    2016-08-01

    In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal Apaired-Aaligned GFRα1-positive spermatogonia and was surrounded by Aaligned GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis. PMID:27255992

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

    PubMed

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

    2015-04-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4'-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4'-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

  19. 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

  20. 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.

  1. Single cell lineage tracing reveals that oriented cell division contributes to trabecular morphogenesis and regional specification

    PubMed Central

    Li, Jingjing; Miao, Lianjie; Shieh, David; Spiotto, Ernest; Li, Jian; Zhou, Bin; Paul, Antoni; Schwartz, Robert J.; Firulli, Anthony B.; Singer, Harold A.; Huang, Guoying; Wu, Mingfu

    2016-01-01

    Summary The cardiac trabeculae are sheet-like structures extending from the myocardium that function to increase surface area. A lack of trabeculation causes embryonic lethality due to compromised cardiac function. To understand the cellular and molecular mechanisms of trabecular formation, we genetically labeled individual cardiomyocytes prior to trabeculation via the brainbow multicolor system, and traced and analyzed the labeled cells during trabeculation by whole-embryo clearing and imaging. The clones derived from labeled single cells displayed four different geometric patterns that are derived from different patterns of oriented cell division (OCD) and migration. Of the four types of clones, the inner, transmural, and mixed clones contributed to trabecular cardiomyocytes. Further studies showed that perpendicular OCD is an extrinsic asymmetric cell division that putatively contributes to trabecular regional specification. Furthermore, N-Cadherin deletion in labeled clones disrupted the clonal patterns. In summary, our data demonstrate that OCD contributes to trabecular morphogenesis and specification. PMID:27052172

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

    PubMed

    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. 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

  4. 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

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

    PubMed

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

    2016-05-10

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

  6. 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

  7. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer.

    PubMed

    Bindea, Gabriela; Mlecnik, Bernhard; Tosolini, Marie; Kirilovsky, Amos; Waldner, Maximilian; Obenauf, Anna C; Angell, Helen; Fredriksen, Tessa; Lafontaine, Lucie; Berger, Anne; Bruneval, Patrick; Fridman, Wolf Herman; Becker, Christoph; Pagès, Franck; Speicher, Michael R; Trajanoski, Zlatko; Galon, Jérôme

    2013-10-17

    The complex interactions between tumors and their microenvironment remain to be elucidated. Combining large-scale approaches, we examined the spatio-temporal dynamics of 28 different immune cell types (immunome) infiltrating tumors. We found that the immune infiltrate composition changed at each tumor stage and that particular cells had a major impact on survival. Densities of T follicular helper (Tfh) cells and innate cells increased, whereas most T cell densities decreased along with tumor progression. The number of B cells, which are key players in the core immune network and are associated with prolonged survival, increased at a late stage and showed a dual effect on recurrence and tumor progression. The immune control relevance was demonstrated in three endoscopic orthotopic colon-cancer mouse models. Genomic instability of the chemokine CXCL13 was a mechanism associated with Tfh and B cell infiltration. CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis correlating with survival. This integrative study reveals the immune landscape in human colorectal cancer and the major hallmarks of the microenvironment associated with tumor progression and recurrence. PMID:24138885

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

    PubMed

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

    2016-05-24

    Cell migration is controlled by various Ca(2+) signals. Local Ca(2+) signals, in particular, have been identified as versatile modulators of cell migration because of their spatiotemporal diversity. However, little is known about how local Ca(2+) signals coordinate between the front and rear regions in directionally migrating cells. Here, we elucidate the spatial role of local Ca(2+) signals in directed cell migration through combinatorial application of an optogenetic toolkit. An optically guided cell migration approach revealed the existence of Ca(2+) sparklets mediated by L-type voltage-dependent Ca(2+) channels in the rear part of migrating cells. Notably, we found that this locally concentrated Ca(2+) influx acts as an essential transducer in establishing a global front-to-rear increasing Ca(2+) gradient. This asymmetrical Ca(2+) gradient is crucial for maintaining front-rear morphological polarity by restricting spontaneous lamellipodia formation in the rear part of migrating cells. Collectively, our findings demonstrate a clear link between local Ca(2+) sparklets and front-rear coordination during directed cell migration. PMID:27190091

  10. Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns

    PubMed Central

    Huang, Cheng-Kuang; Donald, Athene

    2015-01-01

    Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t2 law for radial spreading cells, and a t1 law for lengthwise spreading cells. PMID:25551146

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

    PubMed

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

    2015-01-01

    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. PMID:25762114

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

    PubMed Central

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

    2015-01-01

    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. PMID:25762114

  13. Genome-Wide Profiling of Pluripotent Cells Reveals a Unique Molecular Signature of Human Embryonic Germ Cells

    PubMed Central

    Pashai, Nikta; Hao, Haiping; All, Angelo; Gupta, Siddharth; Chaerkady, Raghothama; De Los Angeles, Alejandro; Gearhart, John D.; Kerr, Candace L.

    2012-01-01

    Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency. PMID:22737227

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

    PubMed

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

    2016-08-25

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

  15. 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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

    Vennin, Claire; Herrmann, David; 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

  19. Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

    PubMed Central

    Nern, Aljoscha; Pfeiffer, Barret D.; Rubin, Gerald M.

    2015-01-01

    We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns. PMID:25964354

  20. 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

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

    PubMed Central

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

    2015-01-01

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

  2. 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

  3. Rapid cell-surface prion protein conversion revealed using a novel cell system

    PubMed Central

    Goold, R.; Rabbanian, S.; Sutton, L.; Andre, R.; Arora, P.; Moonga, J.; Clarke, A.R.; Schiavo, G.; Jat, P.; Collinge, J.; Tabrizi, S.J.

    2011-01-01

    Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrPC). Here we develop a unique cell system in which epitope-tagged PrPC is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrPC, when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrPSc). Using this epitope-tagged PrPSc, we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion. PMID:21505437

  4. 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

    2015-02-01

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

  5. 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

    2016-09-01

    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

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

    PubMed Central

    Cordeiro, Ingrid R.; Lopes, Daiana V.; Abreu, José G.; Carneiro, Katia; Rossi, Maria I. D.; Brito, José M.

    2015-01-01

    ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues. PMID:26319582

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

    PubMed

    Darshan, N; Manonmani, H K

    2016-12-01

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

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

    PubMed Central

    Kort, Remco; Keijser, Bart J; Caspers, Martien PM; Schuren, Frank H; Montijn, Roy

    2008-01-01

    Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability. PMID:19061518

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

    PubMed

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

    2015-10-01

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

  10. 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.

  11. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state

    PubMed Central

    Verfaillie, Annelien; Imrichova, Hana; Atak, Zeynep Kalender; Dewaele, Michael; Rambow, Florian; Hulselmans, Gert; Christiaens, Valerie; Svetlichnyy, Dmitry; Luciani, Flavie; Van den Mooter, Laura; Claerhout, Sofie; Fiers, Mark; Journe, Fabrice; Ghanem, Ghanem-Elias; Herrmann, Carl; Halder, Georg; Marine, Jean-Christophe; Aerts, Stein

    2015-01-01

    Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance. PMID:25865119

  12. Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.

    PubMed

    Simon, Petra; Omokoko, Tana A; Breitkreuz, Andrea; Hebich, Lisa; Kreiter, Sebastian; Attig, Sebastian; Konur, Abdo; Britten, Cedrik M; Paret, Claudia; Dhaene, Karl; Türeci, Özlem; Sahin, Ugur

    2014-12-01

    The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes. PMID:25245536

  13. Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

    PubMed Central

    Niu, Kai-Yang; Liao, Hong-Gang; Zheng, Haimei

    2012-01-01

    The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment. PMID:23287885

  14. 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.

  15. 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.

  16. Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors

    PubMed Central

    Park, Ryan J.; Shen, Hongying; Liu, Lijuan; Liu, Xinran; Ferguson, Shawn M.; De Camilli, Pietro

    2013-01-01

    Summary Dynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors. PMID:24046449

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26483210

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

    PubMed

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

    2016-09-01

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

  19. Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii.

    PubMed

    Yang, Hao; Yang, Ning; Wang, Tai

    2016-03-01

    In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC. PMID:26846354

  20. 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

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

    PubMed

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

    2013-04-10

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

  2. Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells.

    PubMed

    Godin, Antoine G; Rappaz, Benjamin; Potvin-Trottier, Laurent; Kennedy, Timothy E; De Koninck, Yves; Wiseman, Paul W

    2015-08-18

    Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and

  3. Revealing Glycoproteins in the Secretome of MCF-7 Human Breast Cancer Cells

    PubMed Central

    Tan, Aik-Aun; Phang, Wai-Mei; Gopinath, Subash C. B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2015-01-01

    Breast cancer is one of the major issues in the field of oncology, reported with a higher prevalence rate in women worldwide. In attempt to reveal the potential biomarkers for breast cancer, the findings of differentially glycosylated haptoglobin and osteonectin in previous study have drawn our attention towards glycoproteins of secretome from the MCF-7 cancer cell line. In the present study, further analyses were performed on the medium of MCF-7 cells by subjecting it to two-dimensional analyses followed by image analysis in contrast to the medium of human mammary epithelial cells (HMEpC) as a negative control. Carboxypeptidase A4 (CPA4), alpha-1-antitrypsin (AAT), haptoglobin (HP), and HSC70 were detected in the medium of MCF-7, while only CPA4 and osteonectin (ON) were detected in HMEpC medium. In addition, CPA4 was detected as upregulated in the MCF-7 medium. Further analysis by lectin showed that CPA4, AAT, HP, and HSC70 were secreted as N-glycan in the medium of MCF-7, with HP also showing differentially N-glycosylated isoforms. For the HMEpC, only CPA4 was detected as N-glycan. No O-glycan was detected in the medium of HMEpC but MCF-7 expressed O-glycosylated CPA4 and HSC70. All these revealed that glycoproteins could be used as glycan-based biomarkers for the prognosis of breast cancer. PMID:26167486

  4. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation.

    PubMed

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-05-19

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. PMID:27067544

  5. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation

    PubMed Central

    Wang, Maojun; Wang, Pengcheng; Tu, Lili; Zhu, Sitao; Zhang, Lin; Li, Zhonghua; Zhang, Qinghua; Yuan, Daojun; Zhang, Xianlong

    2016-01-01

    Epigenetic modifications are highlighted for their great importance in regulating plant development, but their function associated with single-cell differentiation remains undetermined. Here, we used the cotton fibre, which is the epidermal hair on the cotton ovule, as a model to investigate the regulatory role of DNA methylation in cell differentiation. The level of CHH (H = A, T, or C) DNA methylation level was found to increase during fibre development, accompanied by a decrease in RNA-directed DNA methylation (RdDM). Examination of nucleosome positioning revealed a gradual transition from euchromatin to heterochromatin for chromatin dynamics in developing fibres, which could shape the DNA methylation landscape. The observed increase in DNA methylation in fibres, compared with other ovule tissue, was demonstrated to be mediated predominantly by an active H3K9me2-dependent pathway rather than the RdDM pathway, which was inactive. Furthermore, integrated multi-omics analyses revealed that dynamic DNA methylation played a role in the regulation of lipid biosynthesis and spatio-temporal modulation of reactive oxygen species during fibre differentiation. Our study illustrates two divergent pathways mediating a continuous increase of DNA methylation and also sheds further light on the epigenetic basis for single-cell differentiation in plants. These data and analyses are made available to the wider research community through a comprehensive web portal. PMID:27067544

  6. Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.

    PubMed

    Pageon, Sophie V; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G; Davis, Daniel M

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein. PMID:24209843

  7. 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.

  8. Genetic Manipulations Reveal Dynamic Cell and Gene Functions: Cre-ating a New View of Myogenesis

    PubMed Central

    Hutcheson, David A.; Kardon, Gabrielle

    2010-01-01

    Development of multicellular organisms is temporally and spatially complex. The Cre/loxP and Flp/FRT systems for genetic manipulation in mammals now enable researchers to explicitly examine in vivo the temporal and spatial role of cells and genes during development via cell lineage and ablation studies and conditional gene inactivation and activation. Recently we have used these methods to genetically dissect the role of Pax3+ and Pax7+ progenitor populations and the function of β-catenin, an important regulator of myogenesis, in vertebrate limb myogenesis. Our lineage and ablation studies of Pax3+ and Pax7+ progenitors revealed surprising insights into myogenesis not apparent from Pax3 and Pax7 expression and functional studies. In addition, conditional inactivation and activation of β-catenin in different progenitor populations and their progeny demonstrated that β-catenin plays several cell-autonomous roles in myogenesis. Our studies highlight the hierarchical (i.e. genes versus cells), temporal, and spatial complexity of development and demonstrate that manipulations of both cells and genes will be required to obtain a full understanding of the development of multicellular organisms. PMID:19844163

  9. Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell-wall structure and assembly

    PubMed Central

    Desmarais, Samantha M.; De Pedro, Miguel A.; Cava, Felipe; Huang, Kerwyn Casey

    2013-01-01

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation, and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell-wall synthesis and cell growth. High Performance Liquid Chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques. PMID:23679048

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

    PubMed

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

    2016-01-01

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

  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. 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

  13. Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

    PubMed Central

    Vlasblom, James; Gagarinova, Alla; Phanse, Sadhna; Graham, Chris; Yousif, Fouad; Ding, Huiming; Xiong, Xuejian; Nazarians-Armavil, Anaies; Alamgir, Md; Ali, Mehrab; Pogoutse, Oxana; Pe'er, Asaf; Arnold, Roland; Michaut, Magali; Parkinson, John; Golshani, Ashkan; Whitfield, Chris; Wodak, Shoshana J.; Moreno-Hagelsieb, Gabriel; Greenblatt, Jack F.; Emili, Andrew

    2011-01-01

    As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target. PMID:22125496

  14. 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

  15. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

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

    PubMed

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

    2016-01-01

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

  17. 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

  18. 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.

  19. 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

  20. Methylome, transcriptome, and PPARγ cistrome analyses reveal two epigenetic transitions in fat cells

    PubMed Central

    Takada, Hitomi; Saito, Yutaka; Mituyama, Toutai; Wei, Zong; Yoshihara, Eiji; Jacinto, Sandra; Downes, Michael; Evans, Ronald M; Kida, Yasuyuki S

    2014-01-01

    Although DNA modification is adaptive to extrinsic demands, little is known about epigenetic alterations associated with adipose differentiation and reprogramming. We systematically characterized the global trends of our methylome and transcriptome data with reported PPARγ cistrome data. Our analysis revealed that DNA methylation was altered between induced pluripotent stem cells (iPSCs) and adipose derived stem cells (ADSCs). Surprisingly, DNA methylation was not obviously changed in differentiation from ADSCs to mature fat cells (FatCs). This indicates that epigenetic predetermination of the adipogenic fate is almost established prior to substantial expression of the lineage. Furthermore, the majority of the PPARγ cistrome corresponded to the pre-set methylation profile between ADSCs and FatCs. In contrast to the pre-set model, we found that a subset of PPARγ-binding sites for late-expressing genes such as Adiponectin and Adiponectin receptor2 were differentially methylated independently of the early program. Thus, these analyses identify two types of epigenetic mechanisms that distinguish the pre-set cell fate and later stages of adipose differentiation. PMID:25093444

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

    PubMed Central

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

    2016-01-01

    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. PMID:26893143

  2. 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

  3. Up-regulated expression of Ran reveals its potential role to deltamethrin stress in Kc cells.

    PubMed

    Liu, Wei; Xu, Qin; Chi, Qingping; Hu, Junli; Li, Fengliang; Cheng, Luogen

    2016-05-25

    The GTP-binding nuclear protein Ran has mostly been reported to be an essential player in nuclear transport, chromosome alignment, microtubule dynamics, centrosome duplication, kinetochore attachment of microtubules, nuclear-envelope dynamics, and phagocytosis. However, until now, there has been no report showing the involvement of Ran in DM stress. In this paper, two-dimensional electrophoresis analysis showed that the expression level of Ran in Kc cells in response to DM was higher than that in the control group. In addition, quantitative analysis using real-time PCR revealed that the expression of Ran was obviously up-regulated at various concentrations of DM. Western blot analysis showed that Ran was up-regulated 2.27-fold over the control at 48h. Because we still could not pinpoint whether Ran was actually involved in DM stress reaction, to further verify the role of Ran in stress reaction, RNA interference and cell transfection were utilized. Overexpression of Ran in cells conferred a degree of protection against DM after 72h. Furthermore, interference with Ran significantly decrease cell viability. All of the above findings strongly imply that Ran may participate in the development of stress reaction to DM. Therefore, investigating the possible role of Ran in DM stress will broaden our limited knowledge regarding DM stress inducible genes. PMID:26924245

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

    PubMed

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

    2012-07-01

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

  5. 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

  6. CAFET Algorithm Reveals Wnt/PCP Signature in Lung Squamous Cell Carcinoma

    PubMed Central

    Hu, Yue; Galkin, Anna V.; Wu, Chunlei; Reddy, Venkateshwar; Su, Andrew I.

    2011-01-01

    We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher’s Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis. PMID:22016777

  7. 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.

  8. Molecular processes during fat cell development revealed by gene expression profiling and functional annotation

    PubMed Central

    Hackl, Hubert; Burkard, Thomas Rainer; Sturn, Alexander; Rubio, Renee; Schleiffer, Alexander; Tian, Sun; Quackenbush, John; Eisenhaber, Frank; Trajanoski, Zlatko

    2005-01-01

    Background Large-scale transcription profiling of cell models and model organisms can identify novel molecular components involved in fat cell development. Detailed characterization of the sequences of identified gene products has not been done and global mechanisms have not been investigated. We evaluated the extent to which molecular processes can be revealed by expression profiling and functional annotation of genes that are differentially expressed during fat cell development. Results Mouse microarrays with more than 27,000 elements were developed, and transcriptional profiles of 3T3-L1 cells (pre-adipocyte cells) were monitored during differentiation. In total, 780 differentially expressed expressed sequence tags (ESTs) were subjected to in-depth bioinformatics analyses. The analysis of 3'-untranslated region sequences from 395 ESTs showed that 71% of the differentially expressed genes could be regulated by microRNAs. A molecular atlas of fat cell development was then constructed by de novo functional annotation on a sequence segment/domain-wise basis of 659 protein sequences, and subsequent mapping onto known pathways, possible cellular roles, and subcellular localizations. Key enzymes in 27 out of 36 investigated metabolic pathways were regulated at the transcriptional level, typically at the rate-limiting steps in these pathways. Also, coexpressed genes rarely shared consensus transcription-factor binding sites, and were typically not clustered in adjacent chromosomal regions, but were instead widely dispersed throughout the genome. Conclusions Large-scale transcription profiling in conjunction with sophisticated bioinformatics analyses can provide not only a list of novel players in a particular setting but also a global view on biological processes and molecular networks. PMID:16420668

  9. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure.

    PubMed

    Lu, Zhipeng; Zhang, Qiangfeng Cliff; Lee, Byron; Flynn, Ryan A; Smith, Martin A; Robinson, James T; Davidovich, Chen; Gooding, Anne R; Goodrich, Karen J; Mattick, John S; Mesirov, Jill P; Cech, Thomas R; Chang, Howard Y

    2016-05-19

    RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT. PMID:27180905

  10. 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

  11. 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

  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. PMID:26909555

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

    PubMed Central

    Bazine, Aziz; Fetohi, Mohamed; Tanz, Rachid; Mahfoud, Tarik; Ichou, Mohamed; Errihani, Hassan

    2014-01-01

    Abstract Introduction Cardiac metastases from renal cell carcinoma are very rare. In this report, we describe a case of ventricular metastases in the absence of vena cava or right atrial involvement. Case Report We report the case of a 60-year-old man who had a past history of heavy tobacco intake and well-controlled arterial hypertension. He experienced sudden-onset palpitations, lost consciousness and, as a result, was involved in an accident on the public highway. Cardiac arrhythmia was suspected and, therefore, transthoracic echocardiography was suggested, which revealed a large right ventricular mass. Chest and abdominal computed tomography demonstrated a mass in the right ventricle, but without contiguous vena cava involvement, and a right renal mass related to the probable neoplasm. An ultrasound-guided renal biopsy showed a clear-cell renal cell carcinoma. A bone scan revealed a metastatic bone disease. The patient was started on sunitinib treatment, which was well tolerated. However, approximately 8 months later, reevaluation showed pulmonary metastases. The patient was subsequently started on treatment with everolimus, which, however, was poorly tolerated. Two months later, the patient died due to terminal respiratory insufficiency. Discussion Based on the literature and our observations in this case, targeted antiangiogenic therapy should be considered as a viable therapeutic alternative to metastasectomy for patients with inoperable cardiac metastatic disease as long as there is no baseline systolic or diastolic dysfunction. The case also emphasizes the importance of a thorough history review and physical examination in the workup of patients with syncope. PMID:25232327

  15. 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

  16. 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.

  17. 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. PMID:25710589

  18. 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

  19. 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

  20. Identity and fate of Tbx4-expressing cells reveal developmental cell fate decisions in the allantois, limb, and external genitalia

    PubMed Central

    Naiche, L. A.; Arora, Ripla; Kania, Artur; Lewandoski, Mark; Papaioannou, Virginia E.

    2011-01-01

    T-box gene Tbx4 is critical for the formation of the umbilicus and the initiation of the hindlimb. Previous studies show broad expression in the allantois, hindlimb, lung and proctodeum. We have examined the expression of Tbx4 in detail and used a Tbx4-Cre line to trace the fates of Tbx4-expressing cells. Tbx4 expression and lineage reveal that various distinct appendages, such as the allantois, hindlimb, and external genitalia, all arise from a single mesenchymal expression domain. Additionally, although Tbx4 is associated primarily with the hindlimb, we find two forelimb expression domains. Most notably, we find that, despite the requirement for Tbx4 in allantoic vasculogenesis, the presumptive endothelial cells of the allantois do not express Tbx4 and lineage tracing reveals that the umbilical vasculature never expresses Tbx4. These results imply that endothelial lineages are segregated prior to the onset of vasculogenesis, and demonstrate a role for the peri-vascular tissue in vasculogenesis. PMID:21932311

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

    PubMed Central

    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. PMID:26469858

  2. Proteogenomic analysis reveals unanticipated adaptations of colorectal tumor cells to deficiencies in DNA mismatch repair

    PubMed Central

    Halvey, Patrick J.; Wang, Xiaojing; Wang, Jing; Bhat, Ajaz A.; Dhawan, Punita; Li, Ming; Zhang, Bing; Liebler, Daniel C.; Slebos, Robbert J.C.

    2014-01-01

    Summary A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We employed standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data. Biological replicate cultures yielded highly consistent proteomic inventories with a cumulative total of 6,513 protein groups with a protein FDR of 3.17% across all cell lines. Networks of co-expressed proteins with differential expression based on MMR status revealed impact on protein folding, turnover and transport, on cellular metabolism and on DNA and RNA synthesis and repair. Analysis of variant amino acid sequences suggested higher stability of proteins affected by naturally occurring germline polymorphisms than of proteins affected by somatic protein sequence changes. The data provide evidence for multi-system adaptation to MMR deficiency with a stress response that targets misfolded proteins for degradation through the ubiquitin-dependent proteasome pathway. Enrichment analysis suggested epithelial-to-mesenchymal transition (EMT) in RKO cells, as evidenced by increased mobility and invasion properties compared to SW480. The observed proteomic profiles demonstrate previously unknown consequences of altered DNA repair and provide an expanded basis for mechanistic interpretation of MMR phenotypes. PMID:24247723

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

    PubMed

    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

  4. Morphology-based mammalian stem cell tests reveal potential developmental toxicity of donepezil.

    PubMed

    Lau, Caroline G Y; Marikawa, Yusuke

    2014-11-01

    Various compounds, including therapeutic drugs, can adversely impact the survival and development of embryos in the uterus. Identification of such development-interfering agents is a challenging task, although multi-angle approaches--including the use of in vitro toxicology studies involving embryonic stem cells--should alleviate some of the current difficulties. In the present study, we utilized the in vitro elongation of embryoid bodies (EBs) derived from mouse embryonal carcinoma stem cell line P19C5 as a model of early embryological events, specifically that of gastrulation and axial patterning. From our study, we identified donepezil, a medication indicated for the management of Alzheimer's disease, as a potential developmental toxicant. The extent of P19C5 EB axial elongation was diminished by donepezil in a dose-dependent manner. Although donepezil is a known inhibitor of acetylcholinesterase, interference of elongation was not mediated through this enzyme. Quantitative reverse-transcriptase PCR revealed that donepezil altered the expression pattern of a specific set of developmental regulator genes involved in patterning along the anterior-posterior body axis. When tested in mouse whole embryo culture, donepezil caused morphological abnormalities including impaired somitogenesis. Donepezil also diminished elongation morphogenesis of EBs generated from human embryonic stem cells. These results suggest that donepezil interferes with axial elongation morphogenesis of early embryos by altering the expression pattern of regulators of axial development. PMID:25269881

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

    PubMed

    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

    2016-02-01

    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, or

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

    SciTech Connect

    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. S.; Qian, Weijun

    2009-08-01

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. 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 an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

  7. 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

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

    PubMed

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

    2008-09-25

    The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, 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 the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0-6.5 but not at pH 7 or more. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a non-perturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking. PMID:18818657

  9. 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

  10. Ruptured spleens with expanded marginal zones do not reveal occult B-cell clones.

    PubMed

    Kroft, S H; Singleton, T P; Dahiya, M; Ross, C W; Schnitzer, B; Hsi, E D

    1997-12-01

    An indolent variant of splenic marginal zone lymphoma (SMZL) lacking massive splenomegaly has been described as an incidental finding in spleens removed for rupture or hypersplenism. We studied traumatically ruptured spleens with expanded marginal zones (MZs) to assess the incidence of occult monoclonal B-cell populations in this setting. Ninety-one ruptured or lacerated spleens removed from 1984 to 1995 were classified as to whether they had expanded MZs (> 12 cell layers thick). When available, paraffin-embedded, formalin-fixed tissue from cases with expanded MZs was examined for immunoglobulin heavy chain gene rearrangement by polymerase chain reaction (PCR) and stained for CD20, CD43, and kappa and lambda light chains. Splenectomies were performed for blunt (70 patients) and penetrating (7 patients) trauma, surgical misadventure (13 patients), or spontaneous rupture (1 patient). There were 58 men and 33 women in our study, ranging in age from 17 to 87 years (mean, 40 yr). Average spleen weight was 183 g (range, 44-505 g). Twenty-seven (30%) of 91 patients had expanded MZs. There were no significant differences in age, sex, spleen weight, or reason for excision between those cases with and without MZ expansion. Germinal centers varied from absent to inactive to floridly reactive. Paraffin blocks were available in 24 cases; the 20 with amplifiable DNA were polyclonal by PCR. Follow-up was available for 25 of the 27 patients with expanded MZs (range, 1-85 mo; median, 6 mo); lymphoma did not develop in anyone, although one patient's spleen was morphologically suspicious for lymphoma, showing involvement of red pulp by MZ-type B-cells; PCR revealed a polyclonal pattern. This patient's 3-year follow-up revealed no evidence of lymphoma. Traumatically ruptured spleens with expanded MZs do not seem to harbor occult B-cell clones, as detected by PCR. Although a few cases of incidentally removed spleens have been reported to contain low-stage SMZL, this seems to be an

  11. 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. PMID:24012106

  12. 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

    2015-01-01

    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

  13. 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

    2014-01-01

    Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole exome sequencing of 42 TGCTs to comprehensively study the mutational profile of TGCT. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per megabase [Mb]) as compared to the 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 0.4Mb 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

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

    PubMed

    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

  15. 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

  16. 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. PMID:26502406

  17. 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.

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

    PubMed Central

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

    2014-01-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

  19. STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton-Modulated Lipid Interactions in Living Cells

    PubMed Central

    Mueller, V.; Ringemann, C.; Honigmann, A.; Schwarzmann, G.; Medda, R.; Leutenegger, M.; Polyakova, S.; Belov, V.N.; Hell, S.W.; Eggeling, C.

    2011-01-01

    Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes. PMID:21961591

  20. 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

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

    PubMed

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

    2016-03-01

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

  2. Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells

    PubMed Central

    Tiffert, Teresa

    2011-01-01

    Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca2+-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca2+-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca2+-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca2+ loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase Vmax values much higher than those ever measured by PMCA-mediated Ca2+ extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism. PMID:21948947

  3. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing

    PubMed Central

    Xue, Zhigang; Huang, Kevin; Cai, Chaochao; Cai, Lingbo; Jiang, Chun-yan; Feng, Yun; Liu, Zhenshan; Zeng, Qiao; Cheng, Liming; Sun, Yi E.; Liu, Jia-yin; Horvath, Steve; Fan, Guoping

    2016-01-01

    Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture1–4. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis5,6, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos. PMID:23892778

  4. 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

  5. Principal component analysis of ensemble recordings reveals cell assemblies at high temporal resolution

    PubMed Central

    Benchenane, Karim; Khamassi, Mehdi; Wiener, Sidney I.; Battaglia, Francesco P.

    2009-01-01

    Simultaneous recordings of many single neurons reveals unique insights into network processing spanning the timescale from single spikes to global oscillations. Neurons dynamically self-organize in subgroups of coactivated elements referred to as cell assemblies. Furthermore, these cell assemblies are reactivated, or replayed, preferentially during subsequent rest or sleep episodes, a proposed mechanism for memory trace consolidation. Here we employ Principal Component Analysis to isolate such patterns of neural activity. In addition, a measure is developed to quantify the similarity of instantaneous activity with a template pattern, and we derive theoretical distributions for the null hypothesis of no correlation between spike trains, allowing one to evaluate the statistical significance of instantaneous coactivations. Hence, when applied in an epoch different from the one where the patterns were identified, (e.g. subsequent sleep) this measure allows to identify times and intensities of reactivation. The distribution of this measure provides information on the dynamics of reactivation events: in sleep these occur as transients rather than as a continuous process. PMID:19529888

  6. 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

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

    PubMed

    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

  8. 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.

  9. 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

  10. 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

  11. Gene expression profiling via bioinformatics analysis reveals biomarkers in laryngeal squamous cell carcinoma

    PubMed Central

    GUAN, GUO-FANG; ZHENG, YING; WEN, LIAN-JI; ZHANG, DE-JUN; YU, DUO-JIAO; LU, YAN-QING; ZHAO, YAN; ZHANG, HUI

    2015-01-01

    The present study aimed to identify key genes and relevant microRNAs (miRNAs) involved in laryngeal squamous cell carcinoma (LSCC). The gene expression profiles of LSCC tissue samples were analyzed with various bioinformatics tools. A gene expression data set (GSE51985), including ten laryngeal squamous cell carcinoma (LSCC) tissue samples and ten adjacent non-neoplastic tissue samples, was downloaded from the Gene Expression Omnibus. Differential analysis was performed using software package limma of R. Functional enrichment analysis was applied to the differentially expressed genes (DEGs) using the Database for Annotation, Visualization and Integrated Discovery. Protein-protein interaction (PPI) networks were constructed for the protein products using information from the Search Tool for the Retrieval of Interacting Genes/Proteins. Module analysis was performed using ClusterONE (a software plugin from Cytoscape). MicroRNAs (miRNAs) regulating the DEGs were predicted using WebGestalt. A total of 461 DEGs were identified in LSCC, 297 of which were upregulated and 164 of which were downregulated. Cell cycle, proteasome and DNA replication were significantly over-represented in the upregulated genes, while the ribosome was significantly over-represented in the downregulated genes. Two PPI networks were constructed for the up- and downregulated genes. One module from the upregulated gene network was associated with protein kinase. Numerous miRNAs associated with LSCC were predicted, including miRNA (miR)-25, miR-32, miR-92 and miR-29. In conclusion, numerous key genes and pathways involved in LSCC were revealed, which may aid the advancement of current knowledge regarding the pathogenesis of LSCC. In addition, relevant miRNAs were also identified, which may represent potential biomarkers for use in the diagnosis or treatment of the disease. PMID:25936657

  12. 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. PMID:26820137

  13. Modular expression analysis reveals functional conservation between human Langerhans cells and mouse cross-priming dendritic cells

    PubMed Central

    Artyomov, Maxim N.; Munk, Adiel; Gorvel, Laurent; Korenfeld, Daniel; Cella, Marina; Tung, Thomas

    2015-01-01

    Characterization of functionally distinct dendritic cell (DC) subsets in mice has fueled interest in whether analogous counterparts exist in humans. Transcriptional modules of coordinately expressed genes were used for defining shared functions between the species. Comparing modules derived from four human skin DC subsets and modules derived from the Immunological Genome Project database for all mouse DC subsets revealed that human Langerhans cells (LCs) and the mouse XCR1+CD8α+CD103+ DCs shared the class I–mediated antigen processing and cross-presentation transcriptional modules that were not seen in mouse LCs. Furthermore, human LCs were enriched in a transcriptional signature specific to the blood cross-presenting CD141/BDCA-3+ DCs, the proposed equivalent to mouse CD8α+ DCs. Consistent with our analysis, LCs were highly adept at inducing primary CTL responses. Thus, our study suggests that the function of LCs may not be conserved between mouse and human and supports human LCs as an especially relevant therapeutic target. PMID:25918340

  14. 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

  15. 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

  16. 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.

  17. 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

    2016-02-01

    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

  18. 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

  19. Electrochemical Investigation of a Microbial Solar Cell Reveals a Nonphotosynthetic Biocathode Catalyst

    PubMed Central

    Glaven, Richard H.; Wang, Zheng; Zhou, Jing; Vora, Gary J.; Tender, Leonard M.

    2013-01-01

    Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current–potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria, most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 μm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications. PMID:23603672

  20. Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells.

    PubMed

    Krischek, B; Kasuya, H; Tajima, A; Akagawa, H; Sasaki, T; Yoneyama, T; Ujiie, H; Kubo, O; Bonin, M; Takakura, K; Hori, T; Inoue, I

    2008-07-17

    Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery. Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool. Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation. Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16). Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway "Antigen Presentation" to have the highest upregulation in IA tissue (P=7.3E-10). Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody. Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation. PMID:18538937

  1. Electrochemical investigation of a microbial solar cell reveals a nonphotosynthetic biocathode catalyst.

    PubMed

    Strycharz-Glaven, Sarah M; Glaven, Richard H; Wang, Zheng; Zhou, Jing; Vora, Gary J; Tender, Leonard M

    2013-07-01

    Microbial solar cells (MSCs) are microbial fuel cells (MFCs) that generate their own oxidant and/or fuel through photosynthetic reactions. Here, we present electrochemical analyses and biofilm 16S rRNA gene profiling of biocathodes of sediment/seawater-based MSCs inoculated from the biocathode of a previously described sediment/seawater-based MSC. Electrochemical analyses indicate that for these second-generation MSC biocathodes, catalytic activity diminishes over time if illumination is provided during growth, whereas it remains relatively stable if growth occurs in the dark. For both illuminated and dark MSC biocathodes, cyclic voltammetry reveals a catalytic-current-potential dependency consistent with heterogeneous electron transfer mediated by an insoluble microbial redox cofactor, which was conserved following enrichment of the dark MSC biocathode using a three-electrode configuration. 16S rRNA gene profiling showed Gammaproteobacteria, most closely related to Marinobacter spp., predominated in the enriched biocathode. The enriched biocathode biofilm is easily cultured on graphite cathodes, forms a multimicrobe-thick biofilm (up to 8.2 μm), and does not lose catalytic activity after exchanges of the reactor medium. Moreover, the consortium can be grown on cathodes with only inorganic carbon provided as the carbon source, which may be exploited for proposed bioelectrochemical systems for electrosynthesis of organic carbon from carbon dioxide. These results support a scheme where two distinct communities of organisms develop within MSC biocathodes: one that is photosynthetically active and one that catalyzes reduction of O2 by the cathode, where the former partially inhibits the latter. The relationship between the two communities must be further explored to fully realize the potential for MSC applications. PMID:23603672

  2. Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿ †

    PubMed Central

    Pines, Alex; Kelstrup, Christian D.; Vrouwe, Mischa G.; Puigvert, Jordi C.; Typas, Dimitris; Misovic, Branislav; de Groot, Anton; von Stechow, Louise; van de Water, Bob; Danen, Erik H. J.; Vrieling, Harry; Mullenders, Leon H. F.; Olsen, Jesper V.

    2011-01-01

    Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response. PMID:22006019

  3. Microarray reveals complement components are regulated in the serum-deprived rat retinal ganglion cell line

    PubMed Central

    Khalyfa, Abdelnaby; Chlon, Timothy; Qiang, He; Agarwal, Neeraj

    2007-01-01

    H (CFH), the major inhibitor of the alternative complement pathway is downregulated in serum-deprived RGC-5. CFH protein was detected within RGC-5 cells as well as the rat retina with the aid of immunocytochemistry and confocal microscopy. Conclusions This study was undertaken to generate a genome-wide gene expression profile of RGC-5 after serum deprivation, and to identify candidate and novel genes that may be involved in the signal transduction pathways leading to apoptosis. RGC-5 serum deprivation revealed up-and downregulation in gene expression profiles. The data gathered from this study was the first report that the genes identified in microarray data and validated by real-time RT-PCR may play an important role in RGC-5 cell death. Among the validated genes, C3 and C1s showed significant upregulation of the complement component pathway. The results further indicate that components of the complement pathway are present in neurons of the rat retina. The data indicated that complement factors are likely involved in the pathway leading to ganglion cell death in the serum-deprivation paradigm, which may be similar to the mechanism of cell death in glaucoma. PMID:17356516

  4. Interstitial flows promote an amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model

    PubMed Central

    Huang, Yu Ling; Tung, Chih-kuan; Zheng, Anqi; Kim, Beum Jun; Wu, Mingming

    2015-01-01

    Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumor. Recent in vitro work revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on roles of interstitial flows in modulating heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both an amoeboid and a mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shape shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important roles of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. PMID:26235230

  5. Interstitial flows promote amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model.

    PubMed

    Huang, Yu Ling; Tung, Chih-Kuan; Zheng, Anqi; Kim, Beum Jun; Wu, Mingming

    2015-11-01

    Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumors. Recent in vitro works revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on the role of interstitial flows in modulating the heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both amoeboid and mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shapes shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important role of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. PMID:26235230

  6. A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development.

    PubMed

    Eckelhart, Eva; Warsch, Wolfgang; Zebedin, Eva; Simma, Olivia; Stoiber, Dagmar; Kolbe, Thomas; Rülicke, Thomas; Mueller, Mathias; Casanova, Emilio; Sexl, Veronika

    2011-02-01

    We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell-specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5(f/f) Ncr1-iCreTg animals. Stat5(f/f) Ncr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell-precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2-expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell-mediated tumor control against B16F10-melanoma cells. In contrast, T cell-mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function. PMID:21127177

  7. NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling

    SciTech Connect

    Mace, P.D.; Robinson, H.; Wallez, Y.; Dobaczewska, M. K.; Lee, J. J.; Pasquale, E. B.; Riedl, S. J.

    2011-12-01

    Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas. BCAR3 adopts the Cdc25-homology fold of Ras GTPase exchange factors, but it has a 'closed' conformation incapable of enzymatic activity. The structure of the NSP3-p130Cas complex reveals that this closed conformation is instrumental for interaction of NSP proteins with a focal adhesion-targeting domain present in Cas proteins. This enzyme-to-adaptor conversion enables high-affinity, yet promiscuous, interactions between NSP and Cas proteins and represents an unprecedented mechanistic paradigm linking cellular signaling networks.

  8. Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis.

    PubMed Central

    Van Der Geest, AHM.; Frisch, D. A.; Kemp, J. D.; Hall, T. C.

    1995-01-01

    Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis. PMID:12228659

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

    SciTech Connect

    Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay; Panstruga, Ralph; Uhrig, Joachim; Jones, Alan M

    2011-01-01

    The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} 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.

  10. 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. PMID:25925568

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

    PubMed Central

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

    2015-01-01

    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-N18/19-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. PMID:25925568

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

    NASA Astrophysics Data System (ADS)

    Rand, Barry

    2015-03-01

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

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

    PubMed

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

    2014-01-01

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

  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. Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines.

    PubMed

    Oshima, T; Ikeda, K; Furukawa, M; Ueda, N; Suzuki, H; Takasaka, T

    1996-09-01

    Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC. PMID:8843725

  17. Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

    PubMed Central

    Halmai, Miklos; Frittmann, Orsolya; Szabo, Zoltan; Daraba, Andreea; Gali, Vamsi K.; Balint, Eva; Unk, Ildiko

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well. PMID:27537501

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

    PubMed

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

    2012-03-01

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

  19. Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism

    PubMed Central

    Iwata, Terri N.; Ramírez, Julita A.; Tsang, Mark; Park, Heon; Margineantu, Daciana H.; Hockenbery, David M.

    2016-01-01

    Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism. PMID:27521345

  20. Analysis of leaf development in fugu mutants of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs.

    PubMed

    Ferjani, Ali; Horiguchi, Gorou; Yano, Satoshi; Tsukaya, Hirokazu

    2007-06-01

    In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1-fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways. PMID:17468216

  1. 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

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

    PubMed

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

    2016-07-01

    Mast cells are evolutionarily ancient sentinel cells. Like basophils, mast cells express the high-affinity receptor for immunoglobulin E (IgE) and have been linked to host defense and diverse immune-system-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 shared more overlap with other circulating granulocytes than with mast cells. The derivation of mast-cell and basophil transcriptional signatures underscores their differential capacities to detect environmental signals and influence the inflammatory milieu. PMID:27135604

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

    PubMed

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

    2016-07-26

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

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

    DOE PAGESBeta

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

    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

  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. 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

  7. 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

  8. 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. PMID:24801999

  9. 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

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

    PubMed

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

    2014-02-01

    Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site (a largely decommissioned nuclear production complex) in eastern Washington state, USA, and have been shown to change significantly 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 micro-organisms. 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 extracellular 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, which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic micro-organisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area (in the south-eastern corner of the site) subsurface and biogeochemical shifts associated with Columbia River water intrusion. PMID:24324032

  11. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.

    PubMed

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  12. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes

    PubMed Central

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  13. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    PubMed Central

    2011-01-01

    Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW) proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance. PMID:21884602

  14. Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis.

    PubMed

    Kettman, J R; Soederberg, A; Lefkovits, I

    1986-08-15

    The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation. PMID:3488344

  15. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells.

    PubMed

    Saito, Yo; Fujiwara, Tohru; Ohashi, Keiichi; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Ishizawa, Kenichi; Harigae, Hideo

    2015-01-01

    Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2. PMID:26325290

  16. High-Throughput siRNA Screening to Reveal GATA-2 Upstream Transcriptional Mechanisms in Hematopoietic Cells

    PubMed Central

    Saito, Yo; Fujiwara, Tohru; Ohashi, Keiichi; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Ishizawa, Kenichi; Harigae, Hideo

    2015-01-01

    Hematopoietic stem cells can self-renew and differentiate into all blood cell types. The transcription factor GATA-2 is expressed in both hematopoietic stem and progenitor cells and is essential for cell proliferation, survival, and differentiation. Recently, evidence from studies of aplastic anemia, MonoMAC syndrome, and lung cancer has demonstrated a mechanistic link between GATA-2 and human pathophysiology. GATA-2-dependent disease processes have been extensively analyzed; however, the transcriptional mechanisms upstream of GATA-2 remain less understood. Here, we conducted high-throughput small-interfering-RNA (siRNA) library screening and showed that YN-1, a human erythroleukemia cell line, expressed high levels of GATA-2 following the activation of the hematopoietic-specific 1S promoter. As transient luciferase reporter assay in YN-1 cells revealed the highest promoter activity in the 1S promoter fused with GATA-2 intronic enhancer (+9.9 kb/1S); therefore, we established a cell line capable of stably expressing +9.9 kb/1S-Luciferase. Subsequently, we screened 995 transcription factor genes and revealed that CITED2 acts as a GATA-2 activator in human hematopoietic cells. These results provide novel insights into and further identify the regulatory mechanism of GATA-2. PMID:26325290

  17. A Comparative Mechanical Analysis of Plant and Animal Cells Reveals Convergence across Kingdoms

    PubMed Central

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

    2014-01-01

    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. PMID:25418292

  18. Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus.

    PubMed

    Szabat, Marta; Luciani, Dan S; Piret, James M; Johnson, James D

    2009-04-01

    The enigmatic process of beta-cell maturation has significant implications for diabetes pathogenesis, and potential diabetes therapies. This study examined the dynamics and heterogeneity of insulin and pancreatic duodenal homeobox (Pdx)-1 gene expression in adult beta-cells. Insulin and Pdx1 expression were monitored in human and mouse islet cells and MIN6 cells using a Pdx1-monomeric red fluorescent protein/insulin-enhanced green fluorescent protein dual-reporter lentivirus. The majority of fluorescent cells were highly positive for both Pdx1 and insulin. Cells expressing Pdx1 but little or no insulin (Pdx1(+)/Ins(low)) comprised 15-25% of the total population. Time-lapse imaging demonstrated that Pdx1(+)/Ins(low) primary beta-cells and MIN6 cells could convert to Pdx1(+)/Ins(+) cells without cell division. Genes involved in the mature beta-cell phenotype (Glut2, MafA) were expressed at higher levels in Pdx1(+)/Ins(+) cells relative to Pdx1(+)/Ins(low) cells. Conversely, genes implicated in early beta-cell development (MafB, Nkx2.2) were enriched in Pdx1(+)/Ins(low) cells. Sorted Pdx1(+)/Ins(low) MIN6 cells had a higher replication rate and secreted less insulin relative to double-positive cells. Long-term phenotype tracking of Pdx1(+)/Ins(low) cells showed two groups, one that matured into Pdx1(+)/Ins(+) cells and one that remained immature. These results demonstrate that adult beta-cells pass through distinct maturation states, which is consistent with previously observed heterogeneity in insulin and Pdx1 expression in adult beta-cells. At a given time, a proportion of adult beta-cells share similar characteristics to functionally immature embryonic beta-cell progenitors. The maturation of adult beta-cells recapitulates development in that Pdx1 expression precedes the robust expression of insulin and other mature beta-cell genes. These results have implications for harnessing the maturation process for therapeutic purposes. PMID:19095744

  19. The Metabolic Enzyme ManA Reveals a Link between Cell Wall Integrity and Chromosome Morphology

    PubMed Central

    Elbaz, Maya; Ben-Yehuda, Sigal

    2010-01-01

    Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation. PMID:20862359

  20. Cellular and Molecular Characterization of Human Cardiac Stem Cells Reveals Key Features Essential for Their Function and Safety

    PubMed Central

    Vahdat, Sadaf; Mousavi, Seyed Ahmad; Omrani, Gholamreza; Gholampour, Maziar; Sotoodehnejadnematalahi, Fattah; Ghazizadeh, Zaniar; Gharechahi, Javad

    2015-01-01

    Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT+ cell sorting, clonogenic expansion, and explants culture to isolate c-KIT+ cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT+ and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use. PMID:25867933

  1. Cellular and molecular characterization of human cardiac stem cells reveals key features essential for their function and safety.

    PubMed

    Vahdat, Sadaf; Mousavi, Seyed Ahmad; Omrani, Gholamreza; Gholampour, Maziar; Sotoodehnejadnematalahi, Fattah; Ghazizadeh, Zaniar; Gharechahi, Javad; Baharvand, Hossein; Salekdeh, Ghasem Hosseini; Aghdami, Nasser

    2015-06-15

    Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT(+) cell sorting, clonogenic expansion, and explants culture to isolate c-KIT(+) cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT(+) and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use. PMID:25867933

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

    PubMed

    Neely, Benjamin A; 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

  3. 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

  4. 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. PMID:26901069

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

    PubMed Central

    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. PMID:26901069

  6. Single-cell polyadenylation site mapping reveals 3′ isoform choice variability

    PubMed Central

    Velten, Lars; Anders, Simon; Pekowska, Aleksandra; Järvelin, Aino I; Huber, Wolfgang; Pelechano, Vicent; Steinmetz, Lars M

    2015-01-01

    Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3′ untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3′ isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3′ isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations. PMID:26040288

  7. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    PubMed Central

    Costales, Jaime A; Daily, Johanna P; Burleigh, Barbara A

    2009-01-01

    Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value < 0.01) 24 hours post-invasion. A prominent type I interferon response was observed in each cell type, reflecting a secondary response to secreted cytokine in infected cultures. To identify a core cytokine-independent response in T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at

  8. Cutting edge: An in vivo reporter reveals active B cell receptor signaling in the germinal center.

    PubMed

    Mueller, James; Matloubian, Mehrdad; Zikherman, Julie

    2015-04-01

    Long-lasting Ab responses rely on the germinal center (GC), where B cells bearing high-affinity Ag receptors are selected from a randomly mutated pool to populate the memory and plasma cell compartments. Signaling downstream of the BCR is dampened in GC B cells, raising the possibility that Ag presentation and competition for T cell help, rather than Ag-dependent signaling per se, drive these critical selection events. In this study we use an in vivo reporter of BCR signaling, Nur77-eGFP, to demonstrate that although BCR signaling is reduced among GC B cells, a small population of cells exhibiting GC light zone phenotype (site of Ag and follicular helper T cell encounter) express much higher levels of GFP. We show that these cells exhibit somatic hypermutation, gene expression characteristic of signaling and selection, and undergo BCR signaling in vivo. PMID:25725108

  9. Time-lapse analysis reveals a series of events by which cranial neural crest cells reroute around physical barriers.

    PubMed

    Kulesa, Paul M; Lu, Carole C; Fraser, Scott E

    2005-01-01

    Segmentation is crucial to the development of the vertebrate body plan. Underlying segmentation in the head is further revealed when cranial neural crest cells emerge from even numbered rhombomeres in the hindbrain to form three stereotypical migratory streams that lead to the peripheral branchial arches. To test the role of intrinsic versus extrinsic cues in influencing an individual cell's trajectory, we implanted physical barriers in the chick mesoderm, distal to emerging neural crest cell stream fronts. We analyzed the spatio-temporal dynamics as individual neural crest cells encountered and responded to the barriers, using time-lapse confocal imaging. We find the majority of neural crest cells reach the branchial arch destinations following a repeatable series of events by which the cells overcome the barriers. Even though the lead cells become temporarily blocked by a barrier, cells that follow from behind find a novel pathway around a barrier and become de novo leaders of a new stream. Surprisingly, quantitative analyses of cell trajectories show that cells that encounter an r3 barrier migrate significantly faster but less directly than cells that encounter an r4 barrier, which migrate normally. Interestingly, we also find that cells temporarily blocked by the barrier migrate slightly faster and change direction more often. In addition, we show that cells can be forced to migrate into normally repulsive territory. These results suggest that cranial neural crest cell trajectories are not intrinsically determined, that cells can respond to minor alterations in the environment and re-target a peripheral destination, and that both intrinsic and extrinsic cues are important in patterning. PMID:16254414

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

    PubMed Central

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

    2012-01-01

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

  11. Terminal differentiation of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes.

    PubMed

    Bruder, S P; Caplan, A I

    1990-01-01

    Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the sequence of transitions involved in the osteoblastic cell lineage. These previous data identified distinct cell stages within the osteogenic lineage, but were incomplete. To further refine and extend these observations, additional monoclonal antibodies were generated against the surface of osteogenic cells by immunizing mice with a heterogeneous population of chick embryonic bone cells. Supernatants from growing hybridoma colonies were immunohistochemically screened against frozen sections of stage 35 (day 9.5) chick tibiae. One cell line, SB-5, which secretes an antibody against the surface of osteogenic cells was successfully cloned, stabilized, and immortalized. Studies on the developmental progression of osteogenesis in the embryonic chick tibia reveal that cells within the lineage stages from Pre-Osteoblast to Secretory Osteoblast were never observed to react with antibody SB-5 at any time. By contrast, strong cell surface immunoreactivity was present on mature osteoblastic cells as they became Osteocytes. Furthermore, in cultures of osteogenic cells derived from embryonic calvaria or tibiae, cells possessing the SB-5 antigen on their surface displayed a morphology remarkably similar to that of Osteocytes found in situ. Double immunofluorescent staining of developing chick tibiae with SB-5 and SB-2, a monoclonal antibody directed against the surface of Secretory Osteoblasts, indicates that these cells proceed through an intermediate lineage step before becoming terminally differentiated Osteocytes. This transitory cell state is characterized by the simultaneous cell surface binding of antibodies SB-2 and SB-5, and is referred to as the Osteocytic Osteoblast stage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2202356

  12. Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

    PubMed Central

    Pieler, T; Schreiber, A; Erdmann, V A

    1984-01-01

    Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis. Images PMID:6201825

  13. 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.

  14. 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

  15. Computational Models Reveal a Passive Mechanism for Cell Migration in the Crypt

    PubMed Central

    Dunn, Sara-Jane; Näthke, Inke S.; Osborne, James M.

    2013-01-01

    Cell migration in the intestinal crypt is essential for the regular renewal of the epithelium, and the continued upward movement of cells is a key characteristic of healthy crypt dynamics. However, the driving force behind this migration is unknown. Possibilities include mitotic pressure, active movement driven by motility cues, or negative pressure arising from cell loss at the crypt collar. It is possible that a combination of factors together coordinate migration. Here, three different computational models are used to provide insight into the mechanisms that underpin cell movement in the crypt, by examining the consequence of eliminating cell division on cell movement. Computational simulations agree with existing experimental results, confirming that migration can continue in the absence of mitosis. Importantly, however, simulations allow us to infer mechanisms that are sufficient to generate cell movement, which is not possible through experimental observation alone. The results produced by the three models agree and suggest that cell loss due to apoptosis and extrusion at the crypt collar relieves cell compression below, allowing cells to expand and move upwards. This finding suggests that future experiments should focus on the role of apoptosis and cell extrusion in controlling cell migration in the crypt. PMID:24260407

  16. Surgically resected human tumors reveal the biological significance of the gastric cancer stem cell markers CD44 and CD26

    PubMed Central

    NISHIKAWA, SHIMPEI; KONNO, MASAMITSU; HAMABE, ATSUSHI; HASEGAWA, SHINICHIRO; KANO, YOSHIHIRO; FUKUSUMI, TAKAHITO; SATOH, TAROH; TAKIGUCHI, SHUJI; MORI, MASAKI; DOKI, YUICHIRO; ISHII, HIDESHI

    2015-01-01

    Cancer tissue is maintained by relatively small populations of cancer stem cells (CSCs), which are involved in chemotherapy resistance, recurrence and metastasis. As tumor tissues are comprised of various cells, studies of human clinical samples are important for the characterization of CSCs. In the present study, an expression profiling study was performed in which an anti-cell surface marker antibody-based array platform, a flow cytometry-based cell separation technique and a tumorigenicity analysis in immunodeficient animals were utilized. These approaches revealed that the markers cluster of differentiation (CD)44 and CD26 facilitated the fractionation of surgically resected human gastric cancer (GC) cells into the following subset populations with distinct tumorigenic potentials: Highly tumorigenic CD26+CD44+ cells (6/6 mice formed tumors), moderately tumorigenic CD26+CD44− cells (5/6 mice formed tumors), and weakly or non-tumorigenic CD26−CD44− cells (2/6 mice formed tumors). Furthermore, exposure to 5-fluorouracil significantly increased the proportion of CD26+ cells in vitro. The present study demonstrated that the combined expression of CD26 and CD44 presents a potential marker of human GC stem cells. PMID:26137071

  17. Lineage relationship of CD8(+) T cell subsets is revealed by progressive changes in the epigenetic landscape.

    PubMed

    Crompton, Joseph G; Narayanan, Manikandan; Cuddapah, Suresh; Roychoudhuri, Rahul; Ji, Yun; Yang, Wenjing; Patel, Shashank J; Sukumar, Madhusudhanan; Palmer, Douglas C; Peng, Weiqun; Wang, Ena; Marincola, Francesco M; Klebanoff, Christopher A; Zhao, Keji; Tsang, John S; Gattinoni, Luca; Restifo, Nicholas P

    2016-07-01

    To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8(+) T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8(+) T-cell subsets. Taken together, our results suggest that CD8(+) lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory. PMID:25914936

  18. Lineage relationship of CD8+ T cell subsets is revealed by progressive changes in the epigenetic landscape

    PubMed Central

    Crompton, Joseph G.; Narayanan, Manikandan; Cuddapah, Suresh; Roychoudhuri, Rahul; Ji, Yun; Yang, Wenjing; Patel, Shashank J.; Sukumar, Madhusudhanan; Palmer, Douglas C.; Peng, Weiqun; Wang, Ena; Marincola, Francesco M.; Klebanoff, Christopher A.; Zhao, Keji; Tsang, John S.; Gattinoni, Luca; Restifo, Nicholas P.

    2016-01-01

    To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8+ T-cell subsets. Taken together, our results suggest that CD8+ lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory. PMID:25914936

  19. A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy.

    PubMed

    Hingtgen, Shawn D; Kasmieh, Randa; van de Water, Jeroen; Weissleder, Ralph; Shah, Khalid

    2010-04-01

    Stem cells are promising therapeutic delivery vehicles; however pre-clinical and clinical applications of stem cell-based therapy would benefit significantly from the ability to simultaneously determine therapeutic efficacy and pharmacokinetics of therapies delivered by engineered stem cells. In this study, we engineered and screened numerous fusion variants that contained therapeutic (TRAIL) and diagnostic (luciferase) domains designed to allow simultaneous investigation of multiple events in stem cell-based therapy in vivo. When various stem cell lines were engineered with the optimized molecule, SRL(O)L(2)TR, diagnostic imaging showed marked differences in the levels and duration of secretion between stem cell lines, while the therapeutic activity of the molecule showed the different secretion levels translated to significant variability in tumor cell killing. In vivo, simultaneous diagnostic and therapeutic monitoring revealed that stem cell-based delivery significantly improved pharmacokinetics and anti-tumor effectiveness of the therapy compared to intravenous or intratumoral delivery. As treatment for highly malignant brain tumor xenografts, tracking SRL(O)L(2)TR showed stable stem cell-mediated delivery significantly regressed peripheral and intracranial tumors. Together, the integrated diagnostic and therapeutic properties of SRL(O)L(2)TR answer critical questions necessary for successful utilization of stem cells as novel therapeutic vehicles. PMID:20127797

  20. Epitaxially Grown Collagen Fibrils Reveal Diversity in Contact Guidance Behavior among Cancer Cells

    PubMed Central

    2015-01-01

    Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology and contact guidance behavior of two invasive breast cancer cell lines (MDA-MB-231 and MTLn3 cells). Others have shown that these cells randomly migrate in qualitatively different ways. MDA-MB-231 cells exert large traction forces, tightly adhere to the ECM, and migrate with spindle-shaped morphology and thus adopt a mesenchymal mode of migration. MTLn3 cells exert small traction forces, loosely adhere to the ECM, and migrate with a more rounded morphology and thus adopt an amoeboid mode of migration. As the degree of alignment of type I collagen fibrils increases, cells become more elongated and engage in more directed contact guidance. MDA-MB-231 cells perceive the directional signal of highly aligned type I collagen fibrils with high fidelity, elongating to large extents and migrating directionally. Interestingly, behavior in MTLn3 cells differs. While highly aligned type I collagen fibril patterns facilitate spreading and random migration of MTLn3 cells, they do not support elongation or directed migration. Thus, different contact guidance cues bias cell migration differently and the fidelity of contact guidance is cell type dependent, suggesting that ECM alignment is a permissive cue for contact guidance, but requires a cell to have certain properties to interpret that cue. PMID:25531276

  1. 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.

  2. Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting.

    PubMed Central

    McCaul, T F; Banerjee-Bhatnagar, N; Williams, J C

    1991-01-01

    The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of

  3. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    PubMed Central

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Ramadan, Abdulraouf; Zhang, Qing; Choi, Sung W.; Zhang, Peng; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir; Paczesny, Sophie

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  4. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

    PubMed Central

    van den Broek, Theo; Delemarre, Eveline M.; Janssen, Willemijn J.M.; Nievelstein, Rutger A.J.; Broen, Jasper C.; Tesselaar, Kiki; Borghans, Jose A.M.; Nieuwenhuis, Edward E.S.; Prakken, Berent J.; Mokry, Michal; Jansen, Nicolaas J.G.

    2016-01-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  5. Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.

    PubMed

    van den Broek, Theo; Delemarre, Eveline M; Janssen, Willemijn J M; Nievelstein, Rutger A J; Broen, Jasper C; Tesselaar, Kiki; Borghans, Jose A M; Nieuwenhuis, Edward E S; Prakken, Berent J; Mokry, Michal; Jansen, Nicolaas J G; van Wijk, Femke

    2016-03-01

    The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanding on the identification of truly naive T cells and recent thymic emigrants. PMID:26901814

  6. 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

  7. 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

  8. 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. PMID:26871731

  9. Identification of global gene expression differences between human lens epithelial and cortical fiber cells reveals specific genes and their associated pathways important for specialized lens cell functions

    PubMed Central

    Hawse, John R.; DeAmicis-Tress, Candida; Cowell, Tracy L.; Kantorow, Marc

    2005-01-01

    Purpose: In order to identify specific genes that may play important roles in maintaining the specialized functions of lens epithelial and fiber cells, we have analyzed the global gene expression profiles of these two cell types in the human lens. This analysis will also reveal those genes that are exclusively expressed in the epithelial and cortical fiber cells and those genes that may play important roles in the differentiation of epithelial cells to mature fiber cells. Methods: Oligonucleotide microarray hybridization was used to analyze the expression profiles of 22,215 genes between adult (average age greater than 56 years) human lens epithelial and cortical fiber cells. The expression levels of selected genes were further compared by semi-quantitative RT-PCR and selected genes were functionally clustered into common categories using the EASE bioinformatics software package. Results: Analysis of three separate microarray hybridizations revealed 1,196 transcripts that exhibit increased expression and 1,278 transcripts that exhibit decreased expression at the 2 fold or greater level between lens epithelial cells and cortical fiber cells on all three of the arrays analyzed. Of these, 222 transcripts exhibited increased expression and 135 transcripts exhibited decreased expression by an average of 5 fold or greater levels on all three arrays. Semi-quantitative RT-PCR analysis of 21 randomly selected genes revealed identical expression patterns as those detected by microarray hybridization indicating that the microarray data are accurate. Functional clustering of the identified gene expression patterns using the EASE program revealed a wide variety of biological pathways that exhibited altered expression patterns between the two cell types including mRNA processing, cell adhesion, cell proliferation, translation, protein folding, oxidative phosphorylation, and apoptosis, among others. Conclusions: These data reveal novel and previously identified gene expression

  10. 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. PMID:27521270

  11. Landscape and flux reveal a new global view and physical quantification of mammalian cell cycle

    PubMed Central

    Li, Chunhe; Wang, Jin

    2014-01-01

    Cell cycles, essential for biological function, have been investigated extensively. However, enabling a global understanding and defining a physical quantification of the stability and function of the cell cycle remains challenging. Based upon a mammalian cell cycle gene network, we uncovered the underlying Mexican hat landscape of the cell cycle. We found the emergence of three local basins of attraction and two major potential barriers along the cell cycle trajectory. The three local basins of attraction characterize the G1, S/G2, and M phases. The barriers characterize the G1 and S/G2 checkpoints, respectively, of the cell cycle, thus providing an explanation of the checkpoint mechanism for the cell cycle from the physical perspective. We found that the progression of a cell cycle is determined by two driving forces: curl flux for acceleration and potential barriers for deceleration along the cycle path. Therefore, the cell cycle can be promoted (suppressed), either by enhancing (suppressing) the flux (representing the energy input) or by lowering (increasing) the barrier along the cell cycle path. We found that both the entropy production rate and energy per cell cycle increase as the growth factor increases. This reflects that cell growth and division are driven by energy or nutrition supply. More energy input increases flux and decreases barrier along the cell cycle path, leading to faster oscillations. We also identified certain key genes and regulations for stability and progression of the cell cycle. Some of these findings were evidenced from experiments whereas others lead to predictions and potential anticancer strategies. PMID:25228772

  12. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast

    PubMed Central

    Versari, Cristian; Cinquemani, Eugenio; Ferrari-Trecate, Giancarlo; Hersen, Pascal; Batt, Gregory

    2016-01-01

    Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships) is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation). Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity. PMID:26859137

  13. T Cell Receptor Sequencing Reveals the Clonal Diversity and Overlap of Colonic Effector and FOXP3+ T Cells in Ulcerative Colitis

    PubMed Central

    Lord, James; Chen, Janice; Thirlby, Richard C.; Sherwood, Anna M.; Carlson, Christopher S.

    2015-01-01

    Background & Aims FOXP3+ regulatory T cell (Tregs) prevent inflammation, but are paradoxically increased in ulcerative colitis (UC). Local T cell activation has been hypothesized to account for increased FOXP3 expression in colon lamina propria (LP) T cells. Methods To see if human FOXP3+ LP T cells are an activated fraction of otherwise FOXP3− effector T cells (Teff) and explore their clonal diversity in health and disease, we deep sequenced clonally unique T cell receptor (TCR) hypervariable regions of FOXP3+ and FOXP3− CD4+ T cell subpopulations from inflamed versus non-inflamed colon LP or mesenteric lymph nodes (MLN) of patients with or without UC. Results The clonal diversity of each LP T cell population was no different between patients with versus without UC. Repertoire overlap was only seen between a minority of FOXP3+ and FOXP3− cells, including recently activated CD38+ cells and Th17-like CD161+ Teff, but this repertoire overlap was no different between patients with versus without UC, and was no larger than the overlap between Helios− and Helios+ FOXP3+ cells. Conclusions Thus, at steady state, only a minority of FOXP3+, and particularly Helios+, T cells share a TCR sequence with FOXP3− effector populations in the colon LP, even in UC, revealing distinct clonal origins for LP Tregs and effector T cells in humans. PMID:25437819

  14. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

    PubMed

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-06-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  15. 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.

  16. 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.

  17. Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues.

    PubMed

    Mellott, Adam J; Devarajan, Keerthana; Shinogle, Heather E; Moore, David S; Talata, Zsolt; Laurence, Jennifer S; Forrest, M Laird; Noji, Sumihare; Tanaka, Eiji; Staecker, Hinrich; Detamore, Michael S

    2015-06-01

    The transcription factor atonal homolog 1 (ATOH1) has multiple homologues that are functionally conserved across species and is responsible for the generation of sensory hair cells. To evaluate potential functional differences between homologues, human and mouse ATOH1 (HATH1 and MATH-1, respectively) were nonvirally delivered to human Wharton's jelly cells (hWJCs) for the first time. Delivery of HATH1 to hWJCs demonstrated superior expression of inner ear hair cell markers and characteristics than delivery of MATH-1. Inhibition of HES1 and HES5 signaling further increased the atonal effect. Transfection of hWJCs with HATH1 DNA, HES1 siRNA, and HES5 siRNA displayed positive identification of key hair cell and support cell markers found in the cochlea, as well as a variety of cell shapes, sizes, and features not native to hair cells, suggesting the need for further examination of other cell types induced by HATH1 expression. In the first side-by-side evaluation of HATH1 and MATH-1 in human cells, substantial differences were observed, suggesting that the two atonal homologues may not be interchangeable in human cells, and artificial expression of HATH1 in hWJCs requires further study. In the future, this line of research may lead to engineered systems that would allow for evaluation of drug ototoxicity or potentially even direct therapeutic use. PMID:25760435

  18. Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs.

    PubMed

    Soulika, Marina; Kaushik, Anna-Lila; Mathieu, Benjamin; Lourenço, Raquel; Komisarczuk, Anna Z; Romano, Sebastian Alejo; Jouary, Adrien; Lardennois, Alicia; Tissot, Nicolas; Okada, Shinji; Abe, Keiko; Becker, Thomas S; Kapsimali, Marika

    2016-06-01

    Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly. PMID:27122167

  19. Nonviral Reprogramming of Human Wharton's Jelly Cells Reveals Differences Between ATOH1 Homologues

    PubMed Central

    Mellott, Adam J.; Devarajan, Keerthana; Shinogle, Heather E.; Moore, David S.; Talata, Zsolt; Laurence, Jennifer S.; Forrest, M. Laird; Noji, Sumihare; Tanaka, Eiji; Staecker, Hinrich

    2015-01-01

    The transcription factor atonal homolog 1 (ATOH1) has multiple homologues that are functionally conserved across species and is responsible for the generation of sensory hair cells. To evaluate potential functional differences between homologues, human and mouse ATOH1 (HATH1 and MATH-1, respectively) were nonvirally delivered to human Wharton's jelly cells (hWJCs) for the first time. Delivery of HATH1 to hWJCs demonstrated superior expression of inner ear hair cell markers and characteristics than delivery of MATH-1. Inhibition of HES1 and HES5 signaling further increased the atonal effect. Transfection of hWJCs with HATH1 DNA, HES1 siRNA, and HES5 siRNA displayed positive identification of key hair cell and support cell markers found in the cochlea, as well as a variety of cell shapes, sizes, and features not native to hair cells, suggesting the need for further examination of other cell types induced by HATH1 expression. In the first side-by-side evaluation of HATH1 and MATH-1 in human cells, substantial differences were observed, suggesting that the two atonal homologues may not be interchangeable in human cells, and artificial expression of HATH1 in hWJCs requires further study. In the future, this line of research may lead to engineered systems that would allow for evaluation of drug ototoxicity or potentially even direct therapeutic use. PMID:25760435

  20. 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

  1. 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

  2. Cell shape-dependent shear stress on adherent cells in a micro-physiologic system as revealed by FEM.

    PubMed

    Pfister, C; Bozsak, C; Wolf, P; Demmel, F; Brischwein, M

    2015-05-01

    Flow-induced shear stress on adherent cells leads to biochemical signaling and mechanical responses of the cells. To determine the flow-induced shear stress on adherent cells cultured in a micro-scaled reaction chamber, we developed a suitable finite element method model. The influence of the most important parameters-cell shape, cell density, shear modulus and fluid velocity-was investigated. Notably, the cell shape strongly influences the resulting shear stress. Long and smooth cells undergo lower shear stress than more rounded cells. Changes in the curvature of the cells lead to stress peaks and single cells experience higher shear stress values than cells of a confluent monolayer. The computational results of the fluid flow simulation were validated experimentally. We also analyzed the influence of flow-induced shear stress on the metabolic activity and shape of L929, a mouse fibroblast cell line, experimentally. The results indicate that threshold stress values for continuous flow conditions cannot be transferred to quasi static flow conditions interrupted by short fluid exchange events. PMID:25856467

  3. Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways

    PubMed Central

    Laudanski, Krzysztof; Miller-Graziano, Carol; Xiao, Wenzhong; Mindrinos, Michael N.; Richards, Daniel R.; De, Asit; Moldawer, Lyle L.; Maier, Ronald V.; Bankey, Paul; Baker, Henry V.; Brownstein, Bernard H.; Cobb, J. Perren; Calvano, Steve E.; Davis, Ronald W.; Tompkins, Ronald G.

    2006-01-01

    Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Rα). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease. PMID:17032758

  4. 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.

  5. 3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration

    PubMed Central

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

    2015-01-01

    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. PMID:26137552

  6. 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. PMID:26137552

  7. 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. PMID:26655898

  8. NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.

    PubMed

    Murayama, Emi; Sarris, Milka; Redd, Michael; Le Guyader, Dorothée; Vivier, Catherine; Horsley, Wyatt; Trede, Nikolaus; Herbomel, Philippe

    2015-01-01

    The ontogeny of haematopoietic niches in vertebrates is essentially unknown. Here we show that the stromal cells of the caudal haematopoietic tissue (CHT), the first niche where definitive haematopoietic stem/progenitor cells (HSPCs) home in zebrafish development, derive from the caudal somites through an epithelial-mesenchymal transition (EMT). The resulting stromal cell progenitors accompany the formation of the caudal vein sinusoids, the other main component of the CHT niche, and mature into reticular cells lining and interconnecting sinusoids. We characterize a zebrafish mutant defective in definitive haematopoiesis due to a deficiency in the nascent polypeptide-associated complex alpha subunit (NACA). We demonstrate that the defect resides not in HSPCs but in the CHT niche. NACA-deficient stromal cell progenitors initially develop normally together with the sinusoids, and HSPCs home to the resulting niche, but stromal cell maturation is compromised, leading to a niche that is unable to support HSPC maintenance, expansion and differentiation. PMID:26411530

  9. 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. PMID:27159577

  10. Regulation of Purkinje cell alignment by reelin as revealed with CR-50 antibody.

    PubMed

    Miyata, T; Nakajima, K; Mikoshiba, K; Ogawa, M

    1997-05-15

    Cerebellar Purkinje cells are generated in the ventricular zone, migrate outward, and finally form a monolayer in the cortex. In reeler mice, however, most Purkinje cells cluster abnormally in subcortical areas. Reelin, the candidate reeler gene product recognized by the CR-50 monoclonal antibody, is concentrated in a cortical zone along which Purkinje cells are aligned linearly, implying that it may regulate their alignment. We used an in vitro system and a transplantation approach to analyze the function of Reelin. Explant culture for 7 d of cerebella isolated from wild-type and reeler mice at embryonic day 13 (E13) reproduced in a phenotype-dependent manner the two distinct arrangement patterns (linear vs clustered) of Purkinje cells. Extensive CR-50 binding to wild-type explants converted the linear pattern into a reeler-like, clustered pattern. On the other hand, when reeler explants lacking Reelin were crowned with an artificial layer of Reelin+ granule cells, some Reelin molecules were distributed into a superficial zone of the reeler explants, and Purkinje cells formed a linear pattern along the Reelin-rich overlay. This "rescue" effect was also inhibited by CR-50. Hence, Reelin is involved in the Purkinje cell alignment, and the lack of this activity may explain the malformation in reeler cerebella. We further injected Reelin+ granule cells into the fourth ventricle of E12-13 mice. Extensive incorporation of the injected Reelin+ cells into the ventricular zone, but not of Reelin- cells, forced Purkinje cells of the host cerebella to form an aberrant layer, suggesting that premigratory Purkinje cells may already be responsive to Reelin or Reelin-related signals. PMID:9133383

  11. 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

  12. 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.

  13. 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

    2012-08-01

    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. PMID:22947854

  14. 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. PMID:27453325

  15. A Systematic Analysis Reveals Heterogeneous Changes in the Endocytic Activities of Cancer Cells

    PubMed Central

    Elkin, Sarah R.; Bendris, Nawal; Reis, Carlos R.; Zhou, Yunyun; Xie, Yang; Huffman, Kenneth E.; Minna, John D.; Schmid, Sandra L.

    2016-01-01

    Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non–small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics. PMID:26359453

  16. Metabolomics reveals metabolic changes in male reproductive cells exposed to thirdhand smoke

    DOE PAGESBeta

    Xu, Bo; Chen, Minjian; Yao, Mengmeng; Ji, Xiaoli; Mao, Zhilei; Tang, Wei; Qiao, Shanlei; Schick, Suzaynn F.; Mao, Jian-Hua; Hang, Bo; et al

    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

  17. 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.

  18. Green tea consumption is associated with decreased DNA damage among GSTM1-positive smokers regardless of their hOGG1 genotype.

    PubMed

    Hakim, Iman A; Chow, H-H Sherry; Harris, Robin B

    2008-08-01

    The levels of tobacco-related DNA adducts in human tissues reflect a dynamic process that is dependent on the intensity and time of exposure to tobacco smoke, the metabolic balance between activation of detoxification mechanisms, and the removal of adducts by DNA repair and/or cell turnover. Urinary 8-hydroxydeoxyguanosine (8-OHdG) is probably 1 of the most abundant DNA lesions formed during oxidative stress and is proposed as a sensitive biomarker of the overall oxidative DNA damage and repair. We performed this study to determine whether there were differences in increased oxidative stress susceptibility to smoking within the combined GSTM1 and hOGG1 genotypes and the impact of green tea drinking on this. We completed a Phase II randomized, controlled, 3-arm tea intervention trial to study the effect of high consumption of decaffeinated green or black tea or water on urinary 8-OHdG among heavy smokers and to evaluate the roles of GSTM1 and hOGG1 genotypes as effect modifiers. Assessment of urinary 8-OHdG after adjustment for baseline measurements and other potential confounders revealed a significant effect of green tea consumption (P = 0.001). The change from baseline was significant in all GSTM1-positive smokers regardless of their hOGG1 genotype. Our data show that consumption of 4 cups (960 mL) of tea/d is a feasible and safe approach and was associated with a significant decrease in urinary 8-OHdG among green tea consumers. Our finding also suggests that green tea intervention might be effective in decreasing DNA damage in the subgroup of smokers who are GSTM1 positive regardless of their hOGG1 genotype. PMID:18641208

  19. 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. PMID:23407661

  20. Antibiotic sensitivity pattern in blaNDM-1-positive and carbapenemase-producing Enterobacteriaceae

    PubMed Central

    Mulla, Summaiya; Charan, Jaykaran; Rajdev, Sangita

    2016-01-01

    Background: Some studies published in recent time revealed that many bacteria from Enterobacteriaceae group are multi-antibiotic-resistant because of the production enzymes carbapenemase particularly New Delhi metallo-beta-lactamase encoded by gene called blaNDM-1. Looking at public health importance of this issue there is a need for studies at other centers to confirm or refute published findings. Objectives: This study was designed with the aim of exploring antibiotic resistance in Enterobacteriaceae group of bacteria and also to explore gene and enzyme responsible for it. Materials and Methods: Samples of Enterobacteriaceae were collected from wards and outpatient departments. Antibiotic sensitivity was checked by an automated system (VITEK 2 COMPACT). Carbapenemase production was assessed by Modified Hodge Test. Presence of blaNDM-1 was assessed by polymerase chain reaction. Statistics: Frequency and percentage were used to describe the data. Frequency of sensitivity was compared between carbapenemase producers and noncarbapenemase producers by Fisher's exact test. Results: Forty-seven percent bacteria were found to be producing carbapenemase enzyme. These bacteria were significantly less sensitive to cefoperazone, cefepime, and amikacin. Among carbapenemase-producing organisms, 3% and 6% were resistant to tigecycline and colistin, respectively. Forty percent bacteria were found to be having blaNDM-1 gene. There was a significant difference between blaNDM-1-positive and blaNDM-1-negative for sensitivity toward cefoperazone + sulbactam, imipenem, meropenem, amikacin, tobramycine, ciprofloxacin, and levofloxacin. Conclusion: Presence of carbapenemase enzyme and blaNDM-1 gene is associated with high level of resistance in Enterobacteriaceae group of bacteria and only few antibiotics have good sensitivity for these organisms. PMID:26958516

  1. Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell-Specific Long Noncoding RNAs.

    PubMed

    Tayari, Mina Masoumeh; Winkle, Melanie; Kortman, Gertrud; Sietzema, Jantine; de Jong, Debora; Terpstra, Martijn; Mestdagh, Pieter; Kroese, Frans G M; Visser, Lydia; Diepstra, Arjan; Kok, Klaas; van den Berg, Anke; Kluiver, Joost

    2016-09-01

    Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker. PMID:27423697

  2. 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. PMID:27362860

  3. 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

  4. 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. PMID:18832014

  5. ATTENUATION OF THE GANGLION CELL LAYER IN A PREMATURE INFANT REVEALED WITH HANDHELD SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY

    PubMed Central

    Goldberg, Mara R.; Zakka, Fouad R.; Carroll, Joseph J.

    2016-01-01

    Purpose: To report on subclinical retinal abnormalities shown through handheld spectral domain optical coherence tomography on a premature infant. Methods: Case report. Results: The initial and follow-up exams on a premature infant revealed severely attenuated ganglion cell and nerve fiber layers. There was cystoid macular edema in both eyes at the initial visits, which resolved by the 1-year follow-up. Discussion: Optical coherence tomography can reveal significant retinal abnormalities in premature infants which are not detectable through funduscopic exam. Documenting such findings may be useful for the comprehensive management of vision problems in children with a history of premature birth. PMID:26529438

  6. Cells deficient in base-excision repair reveal cancer hallmarks originating from adjustments to genetic instability

    PubMed Central

    Markkanen, Enni; Fischer, Roman; Ledentcova, Marina; Kessler, Benedikt M.; Dianov, Grigory L.

    2015-01-01

    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. PMID:25800737

  7. 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. PMID:26883363

  8. Revealing the cytoskeletal organization of invasive cancer cells in 3D.

    PubMed

    Geraldo, Sara; Simon, Anthony; Vignjevic, Danijela M

    2013-01-01

    Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D. PMID:24192916

  9. 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

  10. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas.

    PubMed

    Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2016-03-01

    Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

  11. Immunologic profiles of multiple sclerosis treatments reveal shared early B cell alterations

    PubMed Central

    Dooley, James; Pauwels, Ine; Franckaert, Dean; Smets, Ide; Garcia-Perez, Josselyn E.; Hilven, Kelly; Danso-Abeam, Dina; Terbeek, Joanne; Nguyen, Anh T.L.; De Muynck, Louis; Decallonne, Brigitte; Dubois, Bénédicte

    2016-01-01

    Objective: We undertook a systems immunology approach of the adaptive immune system in multiple sclerosis (MS), overcoming tradeoffs between scale and level of detail, in order to identify the immunologic signature of MS and the changes wrought by current immunomodulatory treatments. Methods: We developed a comprehensive flow cytometry platform measuring 38 immunologic cell types in the peripheral blood of 245 individuals in a routine clinical setting. These include patients with MS, untreated or receiving any of 4 current immunomodulatory treatments (interferon-β, glatiramer acetate, natalizumab, or fingolimod), patients with autoimmune thyroid disease, and healthy controls. Results: An increase in memory CD8+ T cells and B cells was observed in untreated patients with MS. Interferon-β and fingolimod induce significant changes upon multiple aspects of the peripheral immune system, with an unexpectedly prominent alteration of B cells. Overall, both treatments push the immune system in different directions, with only 2 significant effects shared across these treatments—an increase in transitional B cells and a decrease in class-switched B cells. We further identified heightened B cell-activating factor (BAFF) levels as regulating this shared B cell pathway. Conclusions: A systems immunology approach established different immunologic profiles induced by current immunomodulatory MS treatments, offering perspectives for personalized medicine. Pathways shared between the immunologic architecture of existing efficacious treatments identify targets for future treatment design. PMID:27231713

  12. Gene expression profiling revealed novel mechanism of action of Taxotere and Furtulon in prostate cancer cells

    PubMed Central

    Li, Yiwei; Hussain, Maha; Sarkar, Sarah H; Eliason, James; Li, Ran; Sarkar, Fazlul H

    2005-01-01

    Background Both Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer. Methods The total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data. Results Taxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents. Conclusions Taxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer. PMID:15656911

  13. High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

    PubMed

    Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

    2016-08-01

    Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. PMID:27085557

  14. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas

    PubMed Central

    Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2016-01-01

    Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

  15. Pluripotent embryonic stem cells and multipotent adult germline stem cells reveal similar transcriptomes including pluripotency-related genes.

    PubMed

    Meyer, S; Nolte, J; Opitz, L; Salinas-Riester, G; Engel, W

    2010-11-01

    DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs). PMID:20624824

  16. Analysis of Cell Cycle Phase Response Captures the Synchronization Phenomena and Reveals a Novel Cell Cycle Network Topology

    NASA Astrophysics Data System (ADS)

    Li, Ying; Lin, Yihan; Scherer, Norbert; Dinner, Aaron

    2011-03-01

    Cell cycle progression requires a succession of temporally-regulated sub-processes, including chromosome replication and cell division, which are each controlled by their own regulatory modules. The modular design of cell cycle regulatory network allows robust environmental responses and evolutionary adaptations. It is emerging that some of the cell cycle modules involve their own autonomous periodic dynamics. As a consequence, the realization of robust coordination among these modules becomes challenging since each module could potentially run out of sync. We believe that an insight into this puzzle resides in the coupling between the contributing regulatory modules. Here, we measured the phase response curve (PRC) of the cell cycle oscillator by driving the expression of a master regulator of the cell cycle in a pulsatile manner and measuring the single cell phase response. We constructed a return map that quantitatively explains the synchronization phenomena that were caused by periodic chemical perturbation. To capture the measured phase response, we derived a minimalist coupled oscillator model that generalizes the basic topology of the cell cycle network. This diode-like coupling suggests that the cell is engineered to ensure complete coordination of constituent events with the cell cycle.

  17. Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation

    PubMed Central

    Kandyba, Eve; Leung, Yvonne; Chen, Yi-Bu; Widelitz, Randall; Chuong, Cheng-Ming; Kobielak, Krzysztof

    2013-01-01

    Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell–dermal papilla-based epithelial–mesenchymal interaction layer and the hair follicle–intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation. PMID:23292934

  18. Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging

    PubMed Central

    Roy, Jeremy; Kim, Bongki; Hill, Eric; Visconti, Pablo; Krapf, Dario; Vinegoni, Claudio; Weissleder, Ralph; Brown, Dennis; Breton, Sylvie

    2016-01-01

    Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections—which we call axiopodia—periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2–ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment. PMID:26868824

  19. 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.

  20. 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

    2016-05-01

    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. PMID:27062923

  1. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos

    PubMed Central

    Petropoulos, Sophie; Edsgärd, Daniel; Reinius, Björn; Deng, Qiaolin; Panula, Sarita Pauliina; Codeluppi, Simone; Plaza Reyes, Alvaro; Linnarsson, Sten; Sandberg, Rickard; Lanner, Fredrik

    2016-01-01

    Summary 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. PMID:27062923

  2. 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

  3. Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging.

    PubMed

    Roy, Jeremy; Kim, Bongki; Hill, Eric; Visconti, Pablo; Krapf, Dario; Vinegoni, Claudio; Weissleder, Ralph; Brown, Dennis; Breton, Sylvie

    2016-01-01

    Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections--which we call axiopodia--periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment. PMID:26868824

  4. Superresolution microscopy reveals nanometer-scale reorganization of inhibitory natural killer cell receptors upon activation of NKG2D.

    PubMed

    Pageon, Sophie V; Cordoba, Shaun-Paul; Owen, Dylan M; Rothery, Stephen M; Oszmiana, Anna; Davis, Daniel M

    2013-07-23

    Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development. PMID:23882121

  5. Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation

    PubMed Central

    Ji, Qinqin; Salomon, Arthur R.

    2015-01-01

    The activation of T-lymphocytes through antigen-mediated T-cell receptor (TCR) clustering is vital in regulating the adaptive-immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperature initiated some of the hallmarks of TCR signaling such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum as well as coalesce T-cell membrane microdomains. The precise mechanism of TCR signaling initiation due to temperature change remains obscure. One critical question is whether signaling initiated by cold treatment of T cells differs from signaling initiated by crosslinking of the T cell receptor. To address this uncertainty, a wide-scale, quantitative mass spectrometry-based phosphoproteomic analysis was performed on T cells stimulated either by temperature shift or through crosslinking of the TCR. Careful statistical comparison between the two stimulations revealed a striking level of identity between the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, at unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns nearly identical to soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. PMID:25839225

  6. Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data

    PubMed Central

    Okada, Hiroki; Ohnuki, Shinsuke; Roncero, Cesar; Konopka, James B.; Ohya, Yoshikazu

    2014-01-01

    The cell wall of budding yeast is a rigid structure composed of multiple components. To thoroughly understand its involvement in morphogenesis, we used the image analysis software CalMorph to quantitatively analyze cell morphology after treatment with drugs that inhibit different processes during cell wall synthesis. Cells treated with cell wall–affecting drugs exhibited broader necks and increased morphological variation. Tunicamycin, which inhibits the initial step of N-glycosylation of cell wall mannoproteins, induced morphologies similar to those of strains defective in α-mannosylation. The chitin synthase inhibitor nikkomycin Z induced morphological changes similar to those of mutants defective in chitin transglycosylase, possibly due to the critical role of chitin in anchoring the β-glucan network. To define the mode of action of echinocandin B, a 1,3-β-glucan synthase inhibitor, we compared the morphology it induced with mutants of Fks1 that contains the catalytic domain for 1,3-β-glucan synthesis. Echinocandin B exerted morphological effects similar to those observed in some fks1 mutants, with defects in cell polarity and reduced glucan synthesis activity, suggesting that echinocandin B affects not only 1,3-β-glucan synthesis, but also another functional domain. Thus our multivariate analyses reveal discrete functions of cell wall components and increase our understanding of the pharmacology of antifungal drugs. PMID:24258022

  7. Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry.

    PubMed

    Pirman, David A; Efuet, Ekem; Ding, Xiao-Ping; Pan, Yong; Tan, Lin; Fischer, Susan M; DuBois, Raymond N; Yang, Peiying

    2013-01-01

    Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of

  8. 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

  9. Automated Analysis of Cell Cycle Phase-Specific DNA Damage Reveals Phase-Specific Differences in Cell Sensitivity to Etoposide.

    PubMed

    Luzhin, Artem V; Velichko, Artem K; Razin, Sergey V; Kantidze, Omar L

    2016-10-01

    The comet assay is one of the most widely used approaches for detecting DNA damage; generally, it provides information on the cell population-averaged level of DNA damage. Here, we present an automatic technique for easy measurement of standard comet characteristics and an annotation of the cell cycle phase of each comet. The approach includes the modified neutral comet assay and a pipeline for CellProfiler software designed to analyze DNA damage-related characteristics and annotate the cell cycle phase of each comet. Using this technique we have performed cell cycle phase-specific analysis of DNA damage induced by the topoisomerase II poison etoposide and have shown that the sensitivity of cells to this drug dramatically differed according to their cell cycle phase. It became evident from our results that the proposed protocol provides important additional information that often remains hidden in a standard comet analysis of an asynchronous cell population. J. Cell. Biochem. 117: 2209-2214, 2016. © 2016 Wiley Periodicals, Inc. PMID:27240930

  10. 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

  11. Superresolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth

    PubMed Central

    Ishitsuka, Yuji; Savage, Natasha; Li, Yiming; Bergs, Anna; Grün, Nathalie; Kohler, Daria; Donnelly, Rebecca; Nienhaus, G. Ulrich; Fischer, Reinhard; Takeshita, Norio

    2015-01-01

    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. PMID:26665168

  12. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    PubMed

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line. PMID:27046137

  13. Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation.

    PubMed

    Roychoudhuri, Rahul; Lefebvre, Francois; Honda, Mitsuo; Pan, Li; Ji, Yun; Klebanoff, Christopher A; Nichols, Carmen N; Fourati, Slim; Hegazy, Ahmed N; Goulet, Jean-Philippe; Gattinoni, Luca; Nabel, Gary J; Gilliet, Michel; Cameron, Mark; Restifo, Nicholas P; Sékaly, Rafick P; Flatz, Lukas

    2015-02-11

    The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy. PMID:25446821

  14. Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity

    PubMed Central

    Small, Eliza C.; Xi, Liqun; Wang, Ji-Ping; Widom, Jonathan; Licht, Jonathan D.

    2014-01-01

    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. PMID:24889621

  15. Lineage Tracing Reveals Distinctive Fates for Mesothelial Cells and Submesothelial Fibroblasts during Peritoneal Injury

    PubMed Central

    Chen, Yi-Ting; Chang, Yu-Ting; Pan, Szu-Yu; Chou, Yu-Hsiang; Chang, Fan-Chi; Yeh, Pei-Ying; Liu, Yuan-Hung; Chiang, Wen-Chih; Chen, Yung-Ming; Wu, Kwan-Dun; Tsai, Tun-Jun; Duffield, Jeremy S.

    2014-01-01

    Fibrosis of the peritoneal cavity remains a serious, life-threatening problem in the treatment of kidney failure with peritoneal dialysis. The mechanism of fibrosis remains unclear partly because the fibrogenic cells have not been identified with certainty. Recent studies have proposed mesothelial cells to be an important source of myofibroblasts through the epithelial–mesenchymal transition; however, confirmatory studies in vivo are lacking. Here, we show by inducible genetic fate mapping that type I collagen–producing submesothelial fibroblasts are specific progenitors of α-smooth muscle actin–positive myofibroblasts that accumulate progressively in models of peritoneal fibrosis induced by sodium hypochlorite, hyperglycemic dialysis solutions, or TGF-β1. Similar genetic mapping of Wilms’ tumor-1–positive mesothelial cells indicated that peritoneal membrane disruption is repaired and replaced by surviving mesothelial cells in peritoneal injury, and not by submesothelial fibroblasts. Although primary cultures of mesothelial cells or submesothelial fibroblasts each expressed α-smooth muscle actin under the influence of TGF-β1, only submesothelial fibroblasts expressed α-smooth muscle actin after induction of peritoneal fibrosis in mice. Furthermore, pharmacologic inhibition of the PDGF receptor, which is expressed by submesothelial fibroblasts but not mesothelial cells, attenuated the peritoneal fibrosis but not the remesothelialization induced by hypochlorite. Thus, our data identify distinctive fates for injured mesothelial cells and submesothelial fibroblasts during peritoneal injury and fibrosis. PMID:24854266

  16. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival.

    PubMed

    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

  17. 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

  18. 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. PMID:26665168

  19. A pharmaco-epistasis strategy reveals a new cell size controlling pathway in yeast

    PubMed Central

    Moretto, Fabien; Sagot, Isabelle; Daignan-Fornier, Bertrand; Pinson, Benoît

    2013-01-01

    Cell size is a complex quantitative trait resulting from interactions between intricate genetic networks and environmental conditions. Here, taking advantage of previous studies that uncovered hundreds of genes affecting budding yeast cell size homeostasis, we performed a wide pharmaco-epistasis analysis using drugs mimicking cell size mutations. Simple epistasis relationship emerging from this approach allowed us to characterize a new cell size homeostasis pathway comprising the sirtuin Sir2, downstream effectors including the large ribosomal subunit (60S) and the transcriptional regulators Swi4 and Swi6. We showed that this Sir2/60S signaling route acts independently of other previously described cell size controlling pathways and may integrate the metabolic status of the cell through NAD+ intracellular concentration. Finally, although Sir2 and the 60S subunits regulate both cell size and replicative aging, we found that there is no clear causal relationship between these two complex traits. This study sheds light on a pathway of >50 genes and illustrates how pharmaco-epistasis applied to yeast offers a potent experimental framework to explore complex genotype/phenotype relationships. PMID:24217298

  20. Representational difference analysis of a committed myeloid progenitor cell line reveals evidence for bilineage potential.

    PubMed

    Lawson, N D; Berliner, N

    1998-08-18

    In this study we have sought to characterize a committed myeloid progenitor cell line in an attempt to isolate general factors that may promote differentiation. We used cDNA representational difference analysis (RDA), which allows analysis of differential gene expression, to compare EML and EPRO cells. We have isolated nine differentially expressed cDNA fragments as confirmed by slot blot, Northern, and PCR analysis. Three of nine sequences appear to be novel whereas the identity of the remaining fragments suggested that the EPRO cell line is multipotent. Among the isolated sequences were eosinophilic, monocytic, and neutrophilic specific genes. Therefore, we tested the ability of EPRO cells to differentiate along multiple myeloid lineages and found that EPRO cells exhibited morphologic maturation into either monocyte/macrophages or neutrophils, but not eosinophils. Furthermore, when EPRO cells were exposed to ATRA, neutrophil specific genes were induced, whereas monocytic markers were induced by phorbol ester treatment. This study highlights the use of cDNA RDA in conjunction with the EML/EPRO cell line to isolate markers associated with macrophage and neutrophil differentiation and establishes the usefulness of this system in the search for factors involved in myeloid commitment. PMID:9707612

  1. Distinct Gene Expression Profiles in Egg and Synergid Cells of Rice as Revealed by Cell Type-Specific Microarrays1[W][OA

    PubMed Central

    Ohnishi, Takayuki; Takanashi, Hideki; Mogi, Mirai; Takahashi, Hirokazu; Kikuchi, Shunsuke; Yano, Kentaro; Okamoto, Takashi; Fujita, Masahiro; Kurata, Nori; Tsutsumi, Nobuhiro

    2011-01-01

    Double fertilization in flowering plants refers to a process in which two sperm cells, carried by the pollen tube, fertilize both the egg and the central cell after their release into a synergid cell of the female gametophyte. The molecular processes by which the female gametophytic cells express their unique functions during fertilization are not well understood. Genes expressed in egg and synergid cells might be important for multiple stages of the plant reproductive process. Here, we profiled genome-wide gene expression in egg and synergid cells in rice (Oryza sativa), a model monocot, using a nonenzymatic cell isolation technique. We found that the expression profiles of the egg and synergid cells were already specified at the micropylar end of the female gametophyte during the short developmental period that comprises the three consecutive mitotic nuclear divisions after megaspore generation. In addition, we identified a large number of genes expressed in the rice egg and synergid cells and characterized these genes using Gene Ontology analysis. The analysis suggested that epigenetic and posttranscriptional regulatory mechanisms are involved in the specification and/or maintenance of these cells. Comparisons between the rice profiles and reported Arabidopsis (Arabidopsis thaliana) profiles revealed that genes enriched in the egg/synergid cell of rice were distinct from those in Arabidopsis. PMID:21106719

  2. A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans.

    PubMed

    Mainpal, Rana; Nance, Jeremy; Yanowitz, Judith L

    2015-10-15

    Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation. PMID:26395476

  3. [Revealing the Cell Structure and Formation of Bamboo with Confocal Raman Microscopy].

    PubMed

    Li, Xiao-li; Zhou, Bin-xiong; Zhang, Yi; Yao, Yan-ming; He, Yong

    2016-02-01

    Parenchyma cell (PAC), transition tissue between parenchyma cell and fiber cell (TC) and fibre cell (FC) of bamboo were studied by confocal Raman microscopy in this paper. Partial least squares regression was applied to establish a quantitative differentiation model for the three types of cells. The result showed that the determination coefficients (R²) of calibration and validation were respectively 0.810 and 0.800, and the root mean square error (RMSE) were respectively 0.323 and 0.332. What's more, three raman bands of 1,095, 1,319 and 1,636 cm⁻¹, verified to the characteristic peaks of pectin, hemicellulose and lignin, were found to be the important bands for the differentiation. Subsequently, these three raman bands were used to establish a multiple linear regression (MLR) model, and the determination coefficients (R²) of calibration and validation of the model were respectively 0.644 and 0.643, and the root mean square error (RMSE) were respectively 0.442 and 0.443. This result showed that there existed obvious difference among the three types of cells in these three raman bands. Finally, the raman spectral signal processed by wavelet transform to eliminate baseline were used to chemical imaging analysis. These results showed a rather large microfibril angle between cellulose fibrils and fibre axis, which contributed to higher modulus and hardness of cells. Hemicellulose and cellulose have similar distribution in the raman chemical image, due to the connection of hemicellulose and cellulose microfiber through hydrogen bond and the closely combination under the action of van der Waals force. The cell corners (CC) and compound middle lamella (CML) were heavily lignified, and a gradual decrease of lignification from the outer layer to the inner layer of the three cells indicate that lignification was first occurred at the CC and CML, and the lignification was not fully completed. PMID:27209741

  4. Characterization of a Null Allelic Mutant of the Rice NAL1 Gene Reveals Its Role in Regulating Cell Division

    PubMed Central

    Jiang, Dan; Fang, Jingjing; Lou, Lamei; Zhao, Jinfeng; Yuan, Shoujiang; Yin, Liang; Sun, Wei; Peng, Lixiang; Guo, Baotai; Li, Xueyong

    2015-01-01

    Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. PMID:25658704

  5. Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos.

    PubMed

    de F Maraschin, Simone; Vennik, Marco; Lamers, Gerda E M; Spaink, Herman P; Wang, Mei

    2005-02-01

    Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure

  6. Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division.

    PubMed

    Jiang, Dan; Fang, Jingjing; Lou, Lamei; Zhao, Jinfeng; Yuan, Shoujiang; Yin, Liang; Sun, Wei; Peng, Lixiang; Guo, Baotai; Li, Xueyong

    2015-01-01

    Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogeneous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. PMID:25658704

  7. Quantitative dynamics of triacylglycerol accumulation in microalgae populations at single-cell resolution revealed by Raman microspectroscopy

    PubMed Central

    2014-01-01

    Background Rapid, real-time and label-free measurement of the cellular contents of biofuel molecules such as triacylglycerol (TAG) in populations at single-cell resolution are important for bioprocess control and understanding of the population heterogeneity. Raman microspectroscopy can directly detect the changes of metabolite profile in a cell and thus can potentially serve these purposes. Results Single-cell Raman spectra (SCRS) of the unicellular oleaginous microalgae Nannochloropsis oceanica from the cultures under nitrogen depletion (TAG-producing condition) and nitrogen repletion (non-TAG-producing condition) were sampled at eight time points during the first 96 hours upon the onset of nitrogen depletion. Single N. oceanica cells were captured by a 532-nm laser and the SCRS were acquired by the same laser within one second per cell. Using chemometric methods, the SCRS were able to discriminate cells between nitrogen-replete and nitrogen-depleted conditions at as early as 6 hours with >93.3% accuracy, and among the eight time points under nitrogen depletion with >90.4% accuracy. Quantitative prediction of TAG content in single cells was achieved and validated via SCRS and liquid chromatography-mass spectrometry (LC-MS) analysis at population level. SCRS revealed the dynamics of heterogeneity in TAG production among cells in each isogenic population. A significant negative correlation between TAG content and lipid unsaturation degree in individual microalgae cells was observed. Conclusions Our results show that SCRS can serve as a label-free and non-invasive proxy for quantitatively tracking and screening cellular TAG content in real-time at single-cell level. Phenotypic comparison of single cells via SCRS should also help investigating the mechanisms of functional heterogeneity within a cellular population. PMID:24716544

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

    PubMed Central

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

    2016-01-01

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

  9. 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. PMID:24489879

  10. Measurement of gene regulation in individual cells reveals rapid switching between promoter states.

    PubMed

    Sepúlveda, Leonardo A; Xu, Heng; Zhang, Jing; Wang, Mengyu; Golding, Ido

    2016-03-11

    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, P(RM). We simultaneously measured the concentration of the lambda repressor CI and the number of messenger RNAs (mRNAs) from P(RM) in individual Escherichia 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

  11. 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

    2016-01-01

    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

  12. Quantifying activity: a new assay reveals T-cell signalling in tiny skin biopsy samples.

    PubMed

    Dornmair, Klaus

    2014-06-01

    Tissue-invasive T cells are observed in many inflammatory dermatological diseases, but in most cases, it is not known how they were attracted, what they might recognize, and to which extent they are activated. Answering these questions is surely essential for understanding pathogeneses of the diseases. In a recent issue of Experimental Dermatology, Smith et al. showed that early signalling events in skin-resident T cells may be investigated by multiplex immunoprecipitation flow cytometry, even if only few T cells are available from skin biopsy samples. This new technology will most likely contribute to elucidating the role of skin-invasive T cells and to understanding the pathology of dermatological diseases. PMID:24665970

  13. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching.

    PubMed

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. PMID:27481325

  14. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-04-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  15. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2014-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone’s expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:24717634

  16. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

    PubMed Central

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

  17. Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Daras, Gerasimos; Rigas, Stamatis

    2015-01-01

    Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2-4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2-4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation. PMID:26042727

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Le Dévédec, Sylvia; Waldman, Yedael Y; Stein, Gideon Y; van de Water, Bob

    2014-01-01

    Utilizing molecular data to derive functional physiological models tailored for specific cancer cells can facilitate the use of individually tailored therapies. To this end we present an approach termed PRIME for generating cell-specific genome-scale metabolic models (GSMMs) based on molecular and phenotypic data. We build >280 models of normal and cancer cell-lines that successfully predict metabolic phenotypes in an individual manner. We utilize this set of cell-specific models to predict drug targets that selectively inhibit cancerous but not normal cell proliferation. The top predicted target, MLYCD, is experimentally validated and the metabolic effects of MLYCD depletion investigated. Furthermore, we tested cell-specific predicted responses to the inhibition of metabolic enzymes, and successfully inferred the prognosis of cancer patients based on their PRIME-derived individual GSMMs. These results lay a computational basis and a counterpart experimental proof of concept for future personalized metabolic modeling applications, enhancing the search for novel selective anticancer therapies. DOI: http://dx.doi.org/10.7554/eLife.03641.001 PMID:25415239

  20. IgH sequences in common variable immune deficiency reveal altered B cell development and selection**

    PubMed Central

    Roskin, Krishna M.; Simchoni, Noa; Liu, Yi; Lee, Ji-Yeun; Seo, Katie; Hoh, Ramona A.; Pham, Tho; Park, Joon H.; Furman, David; Dekker, Cornelia L.; Davis, Mark M.; James, Judith A.; Nadeau, Kari C.; Cunningham-Rundles, Charlotte; Boyd, Scott D.

    2015-01-01

    Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ∼1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity determining region 3 (CDR3). We observed decreased selection against antibodies with long CDR3 regions in memory repertoires and decreased V gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive both from decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. CVID patients also exhibited abnormal clonal expansion of unmutated B cells relative to controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B cell stage and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients. PMID:26311730

  1. 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 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. PMID:24671938

  2. Chromosome conformation maps in fission yeast reveal cell cycle dependent sub nuclear structure

    PubMed Central

    Grand, Ralph S.; Pichugina, Tatyana; Gehlen, Lutz R.; Jones, M. Beatrix; Tsai, Peter; Allison, Jane R.; Martienssen, Robert; O'Sullivan, Justin M.

    2014-01-01

    Successful progression through the cell cycle requires spatial and temporal regulation of gene transcript levels and the number, positions and condensation levels of chromosomes. Here we present a high resolution survey of genome interactions in Schizosaccharomyces pombe using synchronized cells to investigate cell cycle dependent changes in genome organization and transcription. Cell cycle dependent interactions were captured between and within S. pombe chromosomes. Known features of genome organization (e.g. the clustering of telomeres and retrotransposon long terminal repeats (LTRs)) were observed throughout the cell cycle. There were clear correlations between transcript levels and chromosomal interactions between genes, consistent with a role for interactions in transcriptional regulation at specific stages of the cell cycle. In silico reconstructions of the chromosome organization within the S. pombe nuclei were made by polymer modeling. These models suggest that groups of genes with high and low, or differentially regulated transcript levels have preferred positions within the S. pombe nucleus. We conclude that the S. pombe nucleus is spatially divided into functional sub-nuclear domains that correlate with gene activity. The observation that chromosomal interactions are maintained even when chromosomes are fully condensed in M phase implicates genome organization in epigenetic inheritance and bookmarking. PMID:25342201

  3. Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

    NASA Astrophysics Data System (ADS)

    Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

    2011-03-01

    Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

  4. Combining AFM and Acoustic Probes to Reveal Changes in the Elastic Stiffness Tensor of Living Cells

    PubMed Central

    Nijenhuis, Nadja; Zhao, Xuegen; Carisey, Alex; Ballestrem, Christoph; Derby, Brian

    2014-01-01

    Knowledge of how the elastic stiffness of a cell affects its communication with its environment is of fundamental importance for the understanding of tissue integrity in health and disease. For stiffness measurements, it has been customary to quote a single parameter quantity, e.g., Young’s modulus, rather than the minimum of two terms of the stiffness tensor required by elasticity theory. In this study, we use two independent methods (acoustic microscopy and atomic force microscopy nanoindentation) to characterize the elastic properties of a cell and thus determine two independent elastic constants. This allows us to explore in detail how the mechanical properties of cells change in response to signaling pathways that are known to regulate the cell’s cytoskeleton. In particular, we demonstrate that altering the tensioning of actin filaments in NIH3T3 cells has a strong influence on the cell's shear modulus but leaves its bulk modulus unchanged. In contrast, altering the polymerization state of actin filaments influences bulk and shear modulus in a similar manner. In addition, we can use the data to directly determine the Poisson ratio of a cell and show that in all cases studied, it is less than, but very close to, 0.5 in value. PMID:25296302

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

    PubMed

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

    2010-09-01

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

  6. Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B-cell responses.

    PubMed

    Veninga, Henrike; Borg, Ellen G F; Vreeman, Kyle; Taylor, Philip R; Kalay, Hakan; van Kooyk, Yvette; Kraal, Georg; Martinez-Pomares, Luisa; den Haan, Joke M M

    2015-03-01

    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

  7. An Atlas of Network Topologies Reveals Design Principles for Caenorhabditis elegans Vulval Precursor Cell Fate Patterning

    PubMed Central

    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. PMID:26114587

  8. The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry

    PubMed Central

    Zhao, Peng; Schulz, Thomas C.; Sherrer, Eric S.; Weatherly, D. Brent; Robins, Allan J.; Wells, Lance

    2015-01-01

    Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked β-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future. PMID:25367160

  9. The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry.

    PubMed

    Zhao, Peng; Schulz, Thomas C; Sherrer, Eric S; Weatherly, D Brent; Robins, Allan J; Wells, Lance

    2015-01-01

    Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked β-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future. PMID:25367160

  10. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.

    PubMed

    Baines, Christopher P; Kaiser, Robert A; Purcell, Nicole H; Blair, N Scott; Osinska, Hanna; Hambleton, Michael A; Brunskill, Eric W; Sayen, M Richard; Gottlieb, Roberta A; Dorn, Gerald W; Robbins, Jeffrey; Molkentin, Jeffery D

    2005-03-31

    Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-alpha-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death. PMID:15800627

  11. Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

    PubMed Central

    Cyrklaff, Marek; Linaroudis, Alexandros; Boicu, Marius; Chlanda, Petr; Baumeister, Wolfgang; Griffiths, Gareth; Krijnse-Locker, Jacomine

    2007-01-01

    At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, w