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Sample records for lines co-express neuronal

  1. Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

    PubMed

    Yap, Karen; Xiao, Yixin; Friedman, Brad A; Je, H Shawn; Makeyev, Eugene V

    2016-05-10

    Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons. PMID:27134173

  2. Co-expression of non-selective cation channels of the transient receptor potential canonical family in central aminergic neurones.

    PubMed

    Sergeeva, Olga A; Korotkova, Tatiana M; Scherer, Annette; Brown, Ritchie E; Haas, Helmut L

    2003-06-01

    The mammalian transient receptor potential canonical (TRPC) group of channels is a family of Ca2+-permeable cation channels that are activated following receptor-mediated stimulation of different isoforms of phospholipase C. In vitro TRPC proteins can form hetero- or homo-oligomeric channels. We performed single-cell RT-PCR analysis to reveal the co-expression of seven TRPC channels in identified rat aminergic neurones. All serotonergic neurones of the dorsal raphe (DR), the majority of histaminergic (tuberomamillary nucleus; TMN) and dopaminergic cells of the ventral tegmental area (VTA), as well as some GABAergic neurones from the VTA, expressed at least one variant of TRPC channels. No TRPC channel expression was found in the locus coeruleus. In raphe neurones TRPC6 and TRPC5 mRNAs occurred most frequently. In VTA and TMN co-expression of TRPC4 with TRPC5 and TRPC6 with TRPC7 was not found in individual neurones (in contrast to the whole-brain regions). Their co-expression in non-neuronal cells could not be excluded. The neonatal TRPC3 subunit was rarely seen. In DR, but not in the other nuclei studied, the expression of orexin receptors correlated with the expression of TRPC channels. We conclude that several TRPC channel populations exist in individual neurones and that their subunit co-expression pattern is region and cell-type specific. PMID:12787073

  3. Midbrain catecholaminergic neurons co-express α-synuclein and tau in progressive supranuclear palsy

    PubMed Central

    Erro Aguirre, María Elena; Zelaya, María Victoria; Sánchez Ruiz de Gordoa, Javier; Tuñón, María Teresa; Lanciego, José Luis

    2015-01-01

    Objective: To analyze the frequency and distribution of α-synuclein deposits in progressive supranuclear palsy (PSP). Methods: The brains of 25 cases of pathologically confirmed PSP were evaluated with immunohistochemistry for α-synuclein and tau. Multiple immunofluorescent stains were applied to analyze the expression of tau and α-synuclein aggregates in catecholaminergic neurons. Patients’ clinical symptoms were retrospectively recorded. Results: Deposits α-synuclein in the form of typical Lewy bodies (LBs) were only found in two PSP cases (8%) that fulfilled the clinical subtype of PSP known as Richardson’s syndrome (RS). LBs were present in the locus ceruleus (LC), substantia nigra pars compacta (SNc), basal forebrain, amygdala and cingulated cortex in a distribution mimicking that of Parkinson’s disease (PD). Triple-immunolabeling revealed co-expression of α-synuclein and tau proteins in some tyrosine hydroxilase (TH)-positive neurons of the LC and SNc. Conclusions: There is no apparent clinical correlation between the presence of LBs in PSP. Tau protein co-aggregate with α-synuclein in catecholaminergic neurons of PSP brains suggesting a synergistic interaction between the two proteins. This is in keeping with the current view of neurodegenerative disorders as “misfolded protein diseases”. PMID:25814937

  4. Neuropeptide co-expression in hypothalamic kisspeptin neurons of laboratory animals and the human

    PubMed Central

    Skrapits, Katalin; Borsay, Beáta Á.; Herczeg, László; Ciofi, Philippe; Liposits, Zsolt; Hrabovszky, Erik

    2015-01-01

    Hypothalamic peptidergic neurons using kisspeptin (KP) and its co-transmitters for communication are critically involved in the regulation of mammalian reproduction and puberty. This article provides an overview of neuropeptides present in KP neurons, with a focus on the human species. Immunohistochemical studies reveal that large subsets of human KP neurons synthesize neurokinin B, as also shown in laboratory animals. In contrast, dynorphin described in KP neurons of rodents and sheep is found rarely in KP cells of human males and postmenopausal females. Similarly, galanin is detectable in mouse, but not human, KP cells, whereas substance P, cocaine- and amphetamine-regulated transcript and proenkephalin-derived opioids are expressed in varying subsets of KP neurons in humans, but not reported in ARC of other species. Human KP neurons do not contain neurotensin, cholecystokinin, proopiomelanocortin-derivatives, agouti-related protein, neuropeptide Y, somatostatin or tyrosine hydroxylase (dopamine). These data identify the possible co-transmitters of human KP cells. Neurochemical properties distinct from those of laboratory species indicate that humans use considerably different neurotransmitter mechanisms to regulate fertility. PMID:25713511

  5. Localization and distribution of neurons that co-express xeroderma pigmentosum-A and epidermal growth factor receptor within Rosenthal's canal.

    PubMed

    Guthrie, O'neil W

    2015-10-01

    Xeroderma pigmentosum-A (XPA) is a C4-type zinc-finger scaffolding protein that regulates the removal of bulky-helix distorting DNA damage products from the genome. Phosphorylation of serine residues within the XPA protein is associated with improved protection of genomic DNA and cell death resistance. Therefore, kinase signaling is one important mechanism for regulating the protective function of XPA. Previous experiments have shown that spiral ganglion neurons (SGNs) may mobilize XPA as a general stress response to chemical and physical ototoxicants. Therapeutic optimization of XPA via kinase signaling could serve as a means to improve DNA repair capacity within neurons following injury. The kinase signaling activity of the epidermal growth factor receptor (EGFR) has been shown in tumor cell lines to increase the repair of DNA damage products that are primarily repaired by XPA. Such observations suggest that EGFR may regulate the protective function of XPA. However, it is not known whether SGNs in particular or neurons in general could co-express XPA and EGFR. In the current study gene and protein expression of XPA and EGFR were determined from cochlear homogenates. Immunofluorescence assays were then employed to localize neurons expressing both EGFR and XPA within the ganglion. This work was then confirmed with double-immunohistochemistry. Rosenthal's canal served as the reference space in these experiments and design-based stereology was employed in first-order stereology quantification of immunoreactive neurons. The results confirmed that a population of SGNs that constitutively express XPA may also express the EGFR. These results provide the basis for future experiments designed to therapeutically manipulate the EGFR in order to regulate XPA activity and restore gene function in neurons following DNA damage. PMID:26493720

  6. Co-expression of anoctamins in cilia of olfactory sensory neurons.

    PubMed

    Henkel, Bastian; Drose, Daniela R; Ackels, Tobias; Oberland, Sonja; Spehr, Marc; Neuhaus, Eva M

    2015-02-01

    Vertebrates can sense and identify a vast array of chemical cues. The molecular machinery involved in chemodetection and transduction is expressed within the cilia of olfactory sensory neurons. Currently, there is only limited information available on the distribution and density of individual signaling components within the ciliary compartment. Using super-resolution microscopy, we show here that cyclic-nucleotide-gated channels and calcium-activated chloride channels of the anoctamin family are localized to discrete microdomains in the ciliary membrane. In addition to ANO2, a second anoctamin, ANO6, also localizes to ciliary microdomains. This observation, together with the fact that ANO6 and ANO2 co-localize, indicates a role for ANO6 in olfactory signaling. We show that both ANO2 and ANO6 can form heteromultimers and that this heteromerization alters the recombinant channels' physiological properties. Thus, we provide evidence for interaction of ANO2 and ANO6 in olfactory cilia, with possible physiological relevance for olfactory signaling. PMID:25500808

  7. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  8. Co-expression of perforin and granzyme B genes induces apoptosis and inhibits the tumorigenicity of laryngeal cancer cell line Hep-2

    PubMed Central

    Li, Xiu-Ying; Li, Zhi; An, Gui-Jie; Liu, Sha; Lai, Yan-Dong

    2014-01-01

    Granzyme B and perforin, two of the most important components, have shown anticancer properties in various cancers, but their effects in laryngeal cancer remain unexplored. Here we decided to examine the effects of Granzyme B and perforin in Hep-2 cells and clarify the role of perforin and granzyme B in the tumorigenicity of laryngeal cancer cell line. Hep-2 cells were transfected with pVAX1-PIG co-expression vector (comprising perforin and granzyme B genes), and then the growth and apoptosis of these Hep-2 cells were evaluated. The tumorigenicity of Hep-2 cell line co-expressing perforin and granzyme B genes was tested in BALB/c nu/nu mice. We found that the co-expression of perforin and granzyme B genes could obviously inhibit cell focus formation and induce cell apoptosis in Hep-2 cells. Furthermore, after subcutaneous injection of Hep-2 cells transfected with pVAX1-PIG, an extensive delay in tumor growth was observed in BALB/c-nu/nu mice. Moreover, our studies demonstrated that the anticancer activity of perforin and granzyme B was sustainable in vivo as tumor development by inducing cell apoptosis. Taken together, our data indicate that the co-expression of perforin and granzyme B genes exhibits anticancer potential, and hopefully provide potential therapeutic applications in laryngeal cancer. PMID:24696715

  9. A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3*

    PubMed Central

    Azarias, Guillaume; Kruusmägi, Markus; Connor, Siobhan; Akkuratov, Evgeny E.; Liu, Xiao-Li; Lyons, David; Brismar, Hjalmar; Broberger, Christian; Aperia, Anita

    2013-01-01

    Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration ([Na+]i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+]i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na+]i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na+]i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na+]i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function. PMID:23195960

  10. Co-expression of GAD67 and choline acetyltransferase reveals a novel neuronal phenotype in the mouse medulla oblongata.

    PubMed

    Gotts, Jittima; Atkinson, Lucy; Edwards, Ian J; Yanagawa, Yuchio; Deuchars, Susan A; Deuchars, Jim

    2015-12-01

    GABAergic and cholinergic systems play an important part in autonomic pathways. To determine the distribution of the enzymes responsible for the production of GABA and acetylcholine in areas involved in autonomic control in the mouse brainstem, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurones, combined with choline acetyl transferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurones were observed throughout the brainstem. A small number of cells contained both ChAT-IR and GAD67-GFP. Such double labelled cells were observed in the NTS (predominantly in the intermediate and central subnuclei), the area postrema, reticular formation and lateral paragigantocellular nucleus. All ChAT-IR neurones in the area postrema contained GAD67-GFP. Double labelled neurones were not observed in the dorsal vagal motor nucleus, nucleus ambiguus or hypoglossal nucleus. Double labelled ChAT-IR/GAD67-GFP cells in the NTS did not contain neuronal nitric oxide synthase (nNOS) immunoreactivity, whereas those in the reticular formation and lateral paragigantocellular nucleus did. The function of these small populations of double labelled cells is currently unknown, however their location suggests a potential role in integrating signals involved in oromotor behaviours. PMID:26015156

  11. Co-expression of the transcription factors CEH-14 and TTX-1 regulates AFD neuron-specific genes gcy-8 and gcy-18 in C. elegans.

    PubMed

    Kagoshima, Hiroshi; Kohara, Yuji

    2015-03-15

    A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification. PMID:25614239

  12. Co-expression of neuronal intermediate filaments, peripherin and α-internexin in human well-differentiated endocrine neoplasms (carcinoid tumors) of the appendix.

    PubMed

    Ishida, Mitsuaki; Kushima, Ryoji; Brevet, Marie; Chatelain, Denis; Okabe, Hidetoshi

    2008-01-01

    The rectum and appendix are the two major sites of well-differentiated endocrine neoplasms (carcinoid tumors) in the lower gastrointestinal tract. Previously, we reported the consistent expression of peripherin in rectal well-differentiated endocrine neoplasms without metastases. However, its expression has not as yet been examined in appendiceal well-differentiated endocrine neoplasms. The aim of our present study was to clarify whether peripherin, a type III neuronal intermediate filament, and α-internexin, a type IV neuronal intermediate filament, are expressed in appendiceal well-differentiated endocrine neoplasms. Other endocrine markers were also examined and compared with the findings from the rectal well-differentiated endocrine neoplasms. The analyses were carried out by immunohistochemical methods using 12 formalin-fixed and paraffin-embedded appendiceal well-differentiated endocrine neoplasms. In all the neoplasms examined, diffuse immunoreactivity of peripherin was observed. In addition, immunoreactivity of α-internexin, which was frequently co-expressed with peripherin, was found in all appendiceal cases. Chromogranin A and neural cell adhesion molecule expression was found in all appendiceal tumors, and serotonin was also frequently expressed (83%, 10/12 cases). Incidences of the expression of these three markers were much higher in the appendiceal than in the rectal cases. Peripherin expression is a common feature of appendiceal and rectal well-differentiated endocrine neoplasms, but the manner of neural marker expression is different depending on the site of origin. It is uncertain whether the expression of peripherin and/or α-internexin is present in the well-differentiated endocrine neoplasms of other organs; further analysis is required to clarify this issue. PMID:21479396

  13. An expression quantitative trait loci-guided co-expression analysis for constructing regulatory network using a rice recombinant inbred line population.

    PubMed

    Wang, Jia; Yu, Huihui; Weng, Xiaoyu; Xie, Weibo; Xu, Caiguo; Li, Xianghua; Xiao, Jinghua; Zhang, Qifa

    2014-03-01

    The ability to reveal the regulatory architecture of genes at the whole-genome level by constructing a regulatory network is critical for understanding the biological processes and developmental programmes of organisms. Here, we conducted an eQTL-guided function-related co-expression analysis to identify the putative regulators and construct gene regulatory network. We performed an eQTL analysis of 210 recombinant inbred lines (RILs) derived from a cross between two indica rice lines, Zhenshan 97 and Minghui 63, the parents of an elite hybrid, using data obtained by hybridizing RNA samples of flag leaves at the heading stage with Affymetrix whole-genome arrays. Making use of an ultrahigh-density single-nucleotide polymorphism bin map constructed by population sequencing, 13 647 eQTLs for 10 725 e-traits were detected, comprising 5079 cis-eQTLs (37.2%) and 8568 trans-eQTLs (62.8%). The analysis revealed 138 trans-eQTLs hotspots, each of which apparently regulates the expression variations of many genes. Co-expression analysis of functionally related genes within the framework of regulator-target relationships outlined by the eQTLs led to the identification of putative regulators in the system. The usefulness of the strategy was demonstrated with the genes known to be involved in flowering. We also applied this strategy to the analysis of QTLs for yield traits, which also suggested likely candidate genes. eQTL-guided co-expression analysis may provide a promising solution for outlining a framework for the complex regulatory network of an organism. PMID:24420573

  14. Development of recombinant cell line co-expressing mutated Nav1.5, Kir2.1, and hERG for the safety assay of drug candidates.

    PubMed

    Fujii, Masato; Ohya, Susumu; Yamamura, Hisao; Imaizumi, Yuji

    2012-07-01

    To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay. PMID:22498908

  15. Co-expression of GAD67 and choline acetyltransferase in neurons in the mouse spinal cord: A focus on lamina X.

    PubMed

    Gotts, Jittima; Atkinson, Lucy; Yanagawa, Yuchio; Deuchars, Jim; Deuchars, Susan A

    2016-09-01

    Lamina X of the spinal cord is a functionally diverse area with roles in locomotion, autonomic control and processing of mechano and nociceptive information. It is also a neurochemically diverse region. However, the different populations of cells in lamina X remain to be fully characterised. To determine the co-localisation of the enzymes responsible for the production of GABA and acetylcholine (which play major roles in the spinal cord) in lamina X of the adult and juvenile mouse, we used a transgenic mouse expressing green fluorescent protein (GFP) in glutamate decarboxylase 67 (GAD67) neurons, combined with choline acetyltransferase (ChAT) immunohistochemistry. ChAT-immunoreactive (IR) and GAD67-GFP containing neurons were observed in lamina X of both adult and juvenile mice and in both age groups a population of cells containing both ChAT-IR and GAD67-GFP were observed in lumbar, thoracic and cervical spinal cord. Such dual labelled cells were predominantly located ventral to the central canal. Immunohistochemistry for vesicular acetylcholine transporter (VAChT) and GAD67 revealed a small number of double labelled terminals located lateral, dorsolateral and ventrolateral to the central canal. This study therefore describes in detail a population of ChAT-IR/GAD67-GFP neurons predominantly ventral to the central canal of the cervical, thoracic and lumbar spinal cord of adult and juvenile mice. These cells potentially correspond to a sub-population of the cholinergic central canal cluster cells which may play a unique role in controlling spinal cord circuitry. PMID:27378584

  16. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

    NASA Technical Reports Server (NTRS)

    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

  17. LINE-1 Retrotransposons: Mediators of Somatic Variation in Neuronal Genomes?

    PubMed Central

    Singer, Tatjana; McConnell, Michael J.; Marchetto, Maria C.N.; Coufal, Nicole G.; Gage, Fred H.

    2010-01-01

    LINE-1 (L1) elements are retrotransposons that insert extra copies of themselves throughout the genome using a “copy and paste” mechanism. L1s have contributed ~20% to total human genome content and are able to influence chromosome integrity and gene expression upon reinsertion. Recent studies show that L1 elements are active and “jumping” during neuronal differentiation. New somatic L1 insertions may generate “genomic plasticity” in neurons by causing variation in genomic DNA sequences and by altering the transcriptome of individual cells. Thus, L1-induced variation may affect neuronal plasticity and behavior. Here, we discuss potential consequences of L1-induced neuronal diversity and propose that a mechanism generating diversity in the brain could broaden the spectrum of behavioral phenotypes that can originate from any single genome. PMID:20471112

  18. An on-line archive of reconstructed hippocampal neurons.

    PubMed

    Cannon, R C; Turner, D A; Pyapali, G K; Wheal, H V

    1998-10-01

    We have developed an on-line archive of neuronal geometry to encourage the use of realistic dendritic structures in morphometry and for neuronal modeling, located at web address www.neuro.soton.ac.uk. Initially we have included full three-dimensional representations of 87 neurons from the hippocampus, obtained following intracellular staining with biocytin and reconstruction using Neurolucida. The archive system includes a structure editor for correcting any departures from valid branching geometry and which allows simple errors in the digitisation to be corrected. The editor employs a platform-independent file format which enforces the constraints that there should be no isolated branches and no closed loops. It also incorporates software for interconversion between the archive format and those used by various neuronal reconstruction and modelling packages. The raw data from digitisation software can be included in the archive as well as edited reconstructions and any further information available. Cross-referenced tables and indexes are updated automatically and are sorted according to a number of fields including the cell type, contributor, submission date and published reference. Both the archive and the structure editor should facilitate the quantitative use of full three-dimensional reconstructions of neurons from the hippocampus and other brain regions. PMID:9821633

  19. MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

    PubMed Central

    Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

    2015-01-01

    The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

  20. Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

    PubMed Central

    Tonazzini, Ilaria; Pellegrini, Monica; Pellegrino, Mario; Cecchini, Marco

    2014-01-01

    Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-µm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells. PMID:24501675

  1. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

    PubMed Central

    Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

    2015-01-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  2. Copper delivery to the CNS by CuATSM effectively treats motor neuron disease in SOD(G93A) mice co-expressing the Copper-Chaperone-for-SOD.

    PubMed

    Williams, Jared R; Trias, Emiliano; Beilby, Pamela R; Lopez, Nathan I; Labut, Edwin M; Bradford, C Samuel; Roberts, Blaine R; McAllum, Erin J; Crouch, Peter J; Rhoads, Timothy W; Pereira, Cliff; Son, Marjatta; Elliott, Jeffrey L; Franco, Maria Clara; Estévez, Alvaro G; Barbeito, Luis; Beckman, Joseph S

    2016-05-01

    Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice. PMID:26826269

  3. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines.

    PubMed

    Gibson, Gary E; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis T; Zhang, Sheng

    2015-07-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl-CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. Reversible post-translation modifications of proteins are common and may regulate many processes. Succinylation of proteins occurs and causes large changes in the structure of proteins. However, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remains unknown. The results demonstrate that the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) can succinylate multiple mitochondrial proteins and alter their function. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  4. On-line, voluntary control of human temporal lobe neurons

    PubMed Central

    Cerf, Moran; Thiruvengadam, Nikhil; Mormann, Florian; Kraskov, Alexander; Quiroga, Rodrigo Quian; Koch, Christof; Fried, Itzhak

    2010-01-01

    Daily life continually confronts us with an exuberance of external, sensory stimuli competing with a rich stream of internal deliberations, plans and ruminations. The brain must select one or more of these for further processing. How this competition is resolved across multiple sensory and cognitive regions is not known; nor is it clear how internal thoughts and attention regulate this competition1–4. Recording from single neurons in patients implanted with intracranial electrodes for clinical reasons5–9, here we demonstrate that humans can regulate the activity of their neurons in the medial temporal lobe (MTL) to alter the outcome of the contest between external images and their internal representation. Subjects looked at a hybrid superposition of two images representing familiar individuals, landmarks, objects or animals and had to enhance one image at the expense of the other, competing one. Simultaneously, the spiking activity of their MTL neurons in different subregions and hemispheres was decoded in real time to control the content of the hybrid. Subjects reliably regulated, often on the first trial, the firing rate of their neurons, increasing the rate of some while simultaneously decreasing the rate of others. They did so by focusing onto one image, which gradually became clearer on the computer screen in front of their eyes, and thereby overriding sensory input. On the basis of the firing of these MTL neurons, the dynamics of the competition between visual images in the subject's mind was visualized on an external display. PMID:20981100

  5. Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

    PubMed Central

    Thompson, Alex F.; Crowe, Megan E.; Lieven, Christopher J.; Levin, Leonard A.

    2015-01-01

    Purpose RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype. Methods 661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology. Results Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested. Conclusions 661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology. PMID:26684837

  6. Morphology and Intrinsic Excitability of Regenerating Sensory and Motor Neurons Grown on a Line Micropattern

    PubMed Central

    Benzina, Ouafa; Cloitre, Thierry; Martin, Marta; Raoul, Cédric; Gergely, Csilla; Scamps, Frédérique

    2014-01-01

    Axonal regeneration is one of the greatest challenges in severe injuries of peripheral nerve. To provide the bridge needed for regeneration, biological or synthetic tubular nerve constructs with aligned architecture have been developed. A key point for improving axonal regeneration is assessing the effects of substrate geometry on neuronal behavior. In the present study, we used an extracellular matrix-micropatterned substrate comprising 3 µm wide lines aimed to physically mimic the in vivo longitudinal axonal growth of mice peripheral sensory and motor neurons. Adult sensory neurons or embryonic motoneurons were seeded and processed for morphological and electrical activity analyses after two days in vitro. We show that micropattern-guided sensory neurons grow one or two axons without secondary branching. Motoneurons polarity was kept on micropattern with a long axon and small dendrites. The micro-patterned substrate maintains the growth promoting effects of conditioning injury and demonstrates, for the first time, that neurite initiation and extension could be differentially regulated by conditioning injury among DRG sensory neuron subpopulations. The micro-patterned substrate impacts the excitability of sensory neurons and promotes the apparition of firing action potentials characteristic for a subclass of mechanosensitive neurons. The line pattern is quite relevant for assessing the regenerative and developmental growth of sensory and motoneurons and offers a unique model for the analysis of the impact of geometry on the expression and the activity of mechanosensitive channels in DRG sensory neurons. PMID:25329060

  7. Frequency response properties of primary afferent neurons in the posterior lateral line system of larval zebrafish.

    PubMed

    Levi, Rafael; Akanyeti, Otar; Ballo, Aleksander; Liao, James C

    2015-01-15

    The ability of fishes to detect water flow with the neuromasts of their lateral line system depends on the physiology of afferent neurons as well as the hydrodynamic environment. Using larval zebrafish (Danio rerio), we measured the basic response properties of primary afferent neurons to mechanical deflections of individual superficial neuromasts. We used two types of stimulation protocols. First, we used sine wave stimulation to characterize the response properties of the afferent neurons. The average frequency-response curve was flat across stimulation frequencies between 0 and 100 Hz, matching the filtering properties of a displacement detector. Spike rate increased asymptotically with frequency, and phase locking was maximal between 10 and 60 Hz. Second, we used pulse train stimulation to analyze the maximum spike rate capabilities. We found that afferent neurons could generate up to 80 spikes/s and could follow a pulse train stimulation rate of up to 40 pulses/s in a reliable and precise manner. Both sine wave and pulse stimulation protocols indicate that an afferent neuron can maintain their evoked activity for longer durations at low stimulation frequencies than at high frequencies. We found one type of afferent neuron based on spontaneous activity patterns and discovered a correlation between the level of spontaneous and evoked activity. Overall, our results establish the baseline response properties of lateral line primary afferent neurons in larval zebrafish, which is a crucial step in understanding how vertebrate mechanoreceptive systems sense and subsequently process information from the environment. PMID:25355959

  8. Frequency response properties of primary afferent neurons in the posterior lateral line system of larval zebrafish

    PubMed Central

    Levi, Rafael; Akanyeti, Otar; Ballo, Aleksander

    2014-01-01

    The ability of fishes to detect water flow with the neuromasts of their lateral line system depends on the physiology of afferent neurons as well as the hydrodynamic environment. Using larval zebrafish (Danio rerio), we measured the basic response properties of primary afferent neurons to mechanical deflections of individual superficial neuromasts. We used two types of stimulation protocols. First, we used sine wave stimulation to characterize the response properties of the afferent neurons. The average frequency-response curve was flat across stimulation frequencies between 0 and 100 Hz, matching the filtering properties of a displacement detector. Spike rate increased asymptotically with frequency, and phase locking was maximal between 10 and 60 Hz. Second, we used pulse train stimulation to analyze the maximum spike rate capabilities. We found that afferent neurons could generate up to 80 spikes/s and could follow a pulse train stimulation rate of up to 40 pulses/s in a reliable and precise manner. Both sine wave and pulse stimulation protocols indicate that an afferent neuron can maintain their evoked activity for longer durations at low stimulation frequencies than at high frequencies. We found one type of afferent neuron based on spontaneous activity patterns and discovered a correlation between the level of spontaneous and evoked activity. Overall, our results establish the baseline response properties of lateral line primary afferent neurons in larval zebrafish, which is a crucial step in understanding how vertebrate mechanoreceptive systems sense and subsequently process information from the environment. PMID:25355959

  9. NeuroLines: A Subway Map Metaphor for Visualizing Nanoscale Neuronal Connectivity.

    PubMed

    Al-Awami, Ali K; Beyer, Johanna; Strobelt, Hendrik; Kasthuri, Narayanan; Lichtman, Jeff W; Pfister, Hanspeter; Hadwiger, Markus

    2014-12-01

    We present NeuroLines, a novel visualization technique designed for scalable detailed analysis of neuronal connectivity at the nanoscale level. The topology of 3D brain tissue data is abstracted into a multi-scale, relative distance-preserving subway map visualization that allows domain scientists to conduct an interactive analysis of neurons and their connectivity. Nanoscale connectomics aims at reverse-engineering the wiring of the brain. Reconstructing and analyzing the detailed connectivity of neurons and neurites (axons, dendrites) will be crucial for understanding the brain and its development and diseases. However, the enormous scale and complexity of nanoscale neuronal connectivity pose big challenges to existing visualization techniques in terms of scalability. NeuroLines offers a scalable visualization framework that can interactively render thousands of neurites, and that supports the detailed analysis of neuronal structures and their connectivity. We describe and analyze the design of NeuroLines based on two real-world use-cases of our collaborators in developmental neuroscience, and investigate its scalability to large-scale neuronal connectivity data. PMID:26356951

  10. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

    PubMed

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  11. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  12. Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

    NASA Astrophysics Data System (ADS)

    Wieringa, Paul; Tonazzini, Ilaria; Micera, Silvestro; Cecchini, Marco

    2012-07-01

    The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

  13. Development of an Immortalised, Post-Pubertal Gonadotrophon-Releasing Hormone Neuronal Cell Line

    PubMed Central

    Wolfe, A.; Ng, Y.; Divall, S. A.; Singh, S. P.; Radovick, S.

    2016-01-01

    Gonadotrophin-releasing hormone (GnRH) is important in reproduction, although some of the mechanisms for its synthesis and release remain elusive. Progress in understanding the GnRH neurone has been hampered by the limited number and diffuse distribution of the neurone in the mammalian brain. Several stable GnRH-expressing cell lines have been developed using in vivo expression of the simian virus 40 T Antigen (TAg), and they have been helpful for the study of gene expression and neuronal function. However, expression of an immortalising gene may interfere with normal cellular function. We developed a novel GnRH-secreting cell line transgenic mouse model suitable for targeted transformation in post-pubertal mice using a tetracycline-regulated TAg transgene. This clonal cell line, GRT, expresses neuronal markers and GnRH. GRT cells grown in medium containing tetracycline-free serum express increasing mRNA levels of GnRH associated with declining levels of TAg expression. The novelty and ultimately the usefulness of this cell line is that TAg expression, which could affect the GnRH neuronal phenotype, can be regulated by tetracycline. PMID:18624926

  14. Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

    SciTech Connect

    Miyake, Seiji; Kobayashi, Saori; Tsubota, Kazuo; Ozawa, Yoko

    2014-04-04

    Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.

  15. Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells

    PubMed Central

    Sadofsky, Laura R.; Sreekrishna, Koti T.; Lin, Yakang; Schinaman, Renee; Gorka, Kate; Mantri, Yogita; Haught, John Christian; Huggins, Thomas G.; Isfort, Robert J.; Bascom, Charles C.; Morice, Alyn H.

    2014-01-01

    Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1. PMID:24921186

  16. A reconfigurable on-line learning spiking neuromorphic processor comprising 256 neurons and 128K synapses

    PubMed Central

    Qiao, Ning; Mostafa, Hesham; Corradi, Federico; Osswald, Marc; Stefanini, Fabio; Sumislawska, Dora; Indiveri, Giacomo

    2015-01-01

    Implementing compact, low-power artificial neural processing systems with real-time on-line learning abilities is still an open challenge. In this paper we present a full-custom mixed-signal VLSI device with neuromorphic learning circuits that emulate the biophysics of real spiking neurons and dynamic synapses for exploring the properties of computational neuroscience models and for building brain-inspired computing systems. The proposed architecture allows the on-chip configuration of a wide range of network connectivities, including recurrent and deep networks, with short-term and long-term plasticity. The device comprises 128 K analog synapse and 256 neuron circuits with biologically plausible dynamics and bi-stable spike-based plasticity mechanisms that endow it with on-line learning abilities. In addition to the analog circuits, the device comprises also asynchronous digital logic circuits for setting different synapse and neuron properties as well as different network configurations. This prototype device, fabricated using a 180 nm 1P6M CMOS process, occupies an area of 51.4 mm2, and consumes approximately 4 mW for typical experiments, for example involving attractor networks. Here we describe the details of the overall architecture and of the individual circuits and present experimental results that showcase its potential. By supporting a wide range of cortical-like computational modules comprising plasticity mechanisms, this device will enable the realization of intelligent autonomous systems with on-line learning capabilities. PMID:25972778

  17. Am80 induces neuronal differentiation in a human neuroblastoma NH-12 cell line.

    PubMed

    Shiohira, Hideo; Kitaoka, Akira; Shirasawa, Hiromi; Enjoji, Munechika; Nakashima, Manabu

    2010-09-01

    Retinoids including natural vitamin A, its derivatives and synthetic compounds work as transcription factors through the retinoic acid receptors (RAR, RXR). All-trans retinoic acid (ATRA), a family of retinoids, is an internal ligand of RAR and well known as a useful differentiation inducer to treat acute promyelocytic leukemia (APL). ATRA therapy is now established as an initial treatment for APL. Recently, to improve therapeutic potency and reduce adverse effects of ATRA, a novel synthetic selective agonist for RARalpha and beta, Am80, was developed and applied to APL treatment. In this study, we tested whether Am80 was capable of inducing neuronal differentiation in a human neuroblastoma cell line, NH-12 and compared the differentiation effects between Am80 and ATRA. Morphological studies demonstrated that Am80 induced more potent neurite outgrowth and also proved lesser cell toxicity than ATRA. Am80 up-regulated the expression of tropomyosin-related kinase B as well as ATRA. Moreover, Am80 increased the expression of the neuronal marker, growth-associated protein 43. These findings suggest that Am80 induces neuronal differentiation to a greater extent than ATRA and thus may help establishing therapeutic strategies against neuronal degenerative disorders such as Parkinson's disease. PMID:20664956

  18. Generation of Human Embryonic Stem Cell Line Expressing zsGreen in Cholinergic Neurons Using CRISPR/Cas9 System.

    PubMed

    Zhou, Jing; Wang, Chencheng; Zhang, Kunshan; Wang, Yingying; Gong, Xi; Wang, Yanlu; Li, Siguang; Luo, Yuping

    2016-08-01

    Lineage specific human embryonic stem cell (hESC) reporter cell line is a versatile tool for biological studies on real time monitoring of differentiation, physiological and biochemical features of special cell types and pathological mechanism of disease. Here we report the generation of ChAT-zsGreen reporter hESC line that express zsGreen under the control of the choline acetyltransferase (ChAT) promoter using CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 system. We show that the ChAT-zsGreen hESC reporter cell lines retain the features of undifferentiated hESC. After cholinergic neuronal differentiation, cholinergic neurons were clearly labeled with green fluorescence protein (zsGreen). The ChAT-zsGreen reporter hESC lines are invaluable not only for the monitoring cholinergic neuronal differentiation but also for study physiological and biochemical hallmarks of cholinergic neurons. PMID:27113041

  19. Cytotoxicity and genotoxicity of zingiberene on different neuron cell lines in vitro.

    PubMed

    Togar, Basak; Turkez, Hasan; Tatar, Abdulgani; Hacimuftuoglu, Ahmet; Geyikoglu, Fatime

    2015-12-01

    The main objective of this study is to investigate the cytotoxic, genotoxic and antioxidant properties of zingiberene (ZBN) in an in vitro rat brain cell culture study. The cytotoxic effect was determined against the rat neuron and N2a neuroblastoma (N2a-NB) cell lines using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while the antioxidant activity was assessed using the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. The effects on DNA damage were also evaluated in this study by the single cell gel electrophoresis assay. The results indicated that ZBN has an anti-proliferative activity suppressing the proliferation of N2a-NB cells at concentrations over 50 mg L(-1) and neuron cells at concentrations over 150 mg L(-1). In addition, ZBN treatments at higher doses (≤50 mg L(-1)) led to increases of TOS levels in N2a-NB cell cultures. However 25 mg L(-1) of ZBN treatment caused increases of TAC levels in cultured neuron and N2a-NB cell cultures while ZBN at doses of 10-400 mg L(-1) did not increase the number of total damage score in both cell lines. This study clearly indicates that ZBN has a significant potential to be used as a natural anticancer agent in cultured N2a-NBs. PMID:24801579

  20. Mechanistic studies of the toxicity of zinc gluconate in the olfactory neuronal cell line Odora.

    PubMed

    Hsieh, Heidi; Vignesh, Kavitha Subramanian; Deepe, George S; Choubey, Divaker; Shertzer, Howard G; Genter, Mary Beth

    2016-09-01

    Zinc is both an essential and potentially toxic metal. It is widely believed that oral zinc supplementation can reduce the effects of the common cold; however, there is strong clinical evidence that intranasal (IN) zinc gluconate (ZG) gel treatment for this purpose causes anosmia, or the loss of the sense of smell, in humans. Using the rat olfactory neuron cell line, Odora, we investigated the molecular mechanism by which zinc exposure exerts its toxic effects on olfactory neurons. Following treatment of Odora cells with 100 and 200μM ZG for 0-24h, RNA-seq and in silico analyses revealed up-regulation of pathways associated with zinc metal response, oxidative stress, and ATP production. We observed that Odora cells recovered from zinc-induced oxidative stress, but ATP depletion persisted with longer exposure to ZG. ZG exposure increased levels of NLRP3 and IL-1β protein levels in a time-dependent manner, suggesting that zinc exposure may cause an inflammasome-mediated cell death, pyroptosis, in olfactory neurons. PMID:27179668

  1. Evaluation of the toxicity of zinc in the rat olfactory neuronal cell line, Odora.

    PubMed

    Hsieh, H; Amlal, H; Genter, M B

    2015-03-01

    Zinc (Zn) has long been touted as a panacea for common cold. Recently, there has been some controversy over whether an intranasal (IN) zinc gluconate gel, purported to fight colds, causes anosmia, or loss of the sense of smell, in humans. Previous evidence has shown that IN zinc sulfate (ZnSO4) solutions can cause anosmia in humans as well as significant damage to the olfactory epithelium in rodents. Using an in vitro olfactory neuron model (the rat Odora cell line), we tested the hypothesis that Zn toxicity was caused by inhibition of the hydrogen voltage-gated channel 1(HVCN1), leading to acidosis and apoptotic cell death. Following studies to characterize the toxicity of zinc gluconate and ZnSO4, Odora cells were grown on coverslips and loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH in the presence and absence of Zn salts. While we found that HVCN1 is not functional in Odora cells, we found that olfactory neurons in vitro maintain their intracellular pH through a sodium/proton exchanger, specifically the sodium proton antiporter 1. ZnSO4, at nontoxic levels, had no impact on intracellular pH after acute exposure or after 24 h of incubation with the cells. In conclusion, Zn toxicity is not mediated through an acidification of intracellular pH in olfactory neurons in vitro. PMID:24980442

  2. The amyloid precursor protein represses expression of acetylcholinesterase in neuronal cell lines.

    PubMed

    Hicks, David A; Makova, Natalia Z; Gough, Mallory; Parkin, Edward T; Nalivaeva, Natalia N; Turner, Anthony J

    2013-09-01

    The toxic role of amyloid β peptides in Alzheimer's disease is well documented. Their generation is via sequential β- and γ-secretase cleavage of the membrane-bound amyloid precursor protein (APP). Other APP metabolites include the soluble ectodomains sAPPα and sAPPβ and also the amyloid precursor protein intracellular domain (AICD). In this study, we examined whether APP is involved in the regulation of acetylcholinesterase (AChE), which is a key protein of the cholinergic system and has been shown to accelerate amyloid fibril formation and increase their toxicity. Overexpression of the neuronal specific isoform, APP695, in the neuronal cell lines SN56 and SH-SY5Y substantially decreased levels of AChE mRNA, protein, and catalytic activity. Although similar decreases in mRNA levels were observed of the proline-rich anchor of AChE, PRiMA, no changes were seen in mRNA levels of the related enzyme, butyryl-cholinesterase, nor of the high-affinity choline transporter. A γ-secretase inhibitor did not affect AChE transcript levels or enzyme activity in SN56 (APP695) or SH-SY5Y (APP695) cells, showing that regulation of AChE by APP does not require the generation of AICD or amyloid β peptide. Treatment of wild-type SN56 cells with siRNA targeting APP resulted in a significant up-regulation in AChE mRNA levels. Mutagenesis studies suggest that the observed transcriptional repression of AChE is mediated by the E1 region of APP, specifically its copper-binding domain, but not the C-terminal YENTPY motif. In conclusion, AChE is regulated in two neuronal cell lines by APP in a manner independent of the generation of sAPPα, sAPPβ, and AICD. PMID:23897820

  3. Neuronal Nitric Oxide Synthase-Mediated Genotoxicity of 2-Methoxyestradiol in Hippocampal HT22 Cell Line.

    PubMed

    Gorska, Magdalena; Zmijewski, Michal A; Kuban-Jankowska, Alicja; Wnuk, Maciej; Rzeszutek, Iwona; Wozniak, Michal

    2016-09-01

    2-methoxyestradiol, metabolite of 17β-estradiol, is considered a potential anticancer agent, currently investigated in several clinical trials. This natural compound was found to be effective towards great number of cancers, including colon, breast, lung, and osteosarcoma and has been reported to be relatively non-toxic towards non-malignant cells. The aim of the study was to determine the potential neurotoxicity and genotoxicity of 2-methoxyestradiol at physiological and pharmacological relevant concentrations in hippocampal HT22 cell line. Herein, we determined influence of 2-methoxyestradiol on proliferation, inhibition of cell cycle, induction of apoptosis, and DNA damage in the HT22 cells. The study was performed using imaging cytometry and comet assay techniques. Herein, we demonstrated that 2-methoxyestradiol, at pharmacologically and also physiologically relevant concentrations, increases nuclear localization of neuronal nitric oxide synthase. It potentially results in DNA strand breaks and increases in genomic instability in hippocampal HT22 cell line. Thus, we are postulating that naturally occurring 2-methoxyestradiol may be considered a physiological modulator of neuron survival. PMID:26381428

  4. Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

    PubMed Central

    Majumder, Mousumi; Rodriguez-Torres, Mauricio; Torres-Garcia, Jose; Wiebe, Ryan; Timoshenko, Alexander V.; Bhattacharjee, Rabindra N.; Chambers, Ann F.; Lala, Peeyush K.

    2012-01-01

    Introduction and Objectives Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. Results A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic

  5. A Framework for the Comparative Assessment of Neuronal Spike Sorting Algorithms towards More Accurate Off-Line and On-Line Microelectrode Arrays Data Analysis.

    PubMed

    Regalia, Giulia; Coelli, Stefania; Biffi, Emilia; Ferrigno, Giancarlo; Pedrocchi, Alessandra

    2016-01-01

    Neuronal spike sorting algorithms are designed to retrieve neuronal network activity on a single-cell level from extracellular multiunit recordings with Microelectrode Arrays (MEAs). In typical analysis of MEA data, one spike sorting algorithm is applied indiscriminately to all electrode signals. However, this approach neglects the dependency of algorithms' performances on the neuronal signals properties at each channel, which require data-centric methods. Moreover, sorting is commonly performed off-line, which is time and memory consuming and prevents researchers from having an immediate glance at ongoing experiments. The aim of this work is to provide a versatile framework to support the evaluation and comparison of different spike classification algorithms suitable for both off-line and on-line analysis. We incorporated different spike sorting "building blocks" into a Matlab-based software, including 4 feature extraction methods, 3 feature clustering methods, and 1 template matching classifier. The framework was validated by applying different algorithms on simulated and real signals from neuronal cultures coupled to MEAs. Moreover, the system has been proven effective in running on-line analysis on a standard desktop computer, after the selection of the most suitable sorting methods. This work provides a useful and versatile instrument for a supported comparison of different options for spike sorting towards more accurate off-line and on-line MEA data analysis. PMID:27239191

  6. A Framework for the Comparative Assessment of Neuronal Spike Sorting Algorithms towards More Accurate Off-Line and On-Line Microelectrode Arrays Data Analysis

    PubMed Central

    Pedrocchi, Alessandra

    2016-01-01

    Neuronal spike sorting algorithms are designed to retrieve neuronal network activity on a single-cell level from extracellular multiunit recordings with Microelectrode Arrays (MEAs). In typical analysis of MEA data, one spike sorting algorithm is applied indiscriminately to all electrode signals. However, this approach neglects the dependency of algorithms' performances on the neuronal signals properties at each channel, which require data-centric methods. Moreover, sorting is commonly performed off-line, which is time and memory consuming and prevents researchers from having an immediate glance at ongoing experiments. The aim of this work is to provide a versatile framework to support the evaluation and comparison of different spike classification algorithms suitable for both off-line and on-line analysis. We incorporated different spike sorting “building blocks” into a Matlab-based software, including 4 feature extraction methods, 3 feature clustering methods, and 1 template matching classifier. The framework was validated by applying different algorithms on simulated and real signals from neuronal cultures coupled to MEAs. Moreover, the system has been proven effective in running on-line analysis on a standard desktop computer, after the selection of the most suitable sorting methods. This work provides a useful and versatile instrument for a supported comparison of different options for spike sorting towards more accurate off-line and on-line MEA data analysis. PMID:27239191

  7. Generation of human cortical neurons from a new immortal fetal neural stem cell line

    SciTech Connect

    Cacci, E.; Villa, A.; Parmar, M.; Cavallaro, M.; Mandahl, N.; Lindvall, O.; Martinez-Serrano, A.; Kokaia, Z. . E-mail: Zaal.Kokaia@med.lu.se

    2007-02-01

    Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

  8. The neuronal basis of on-line visual control in smooth pursuit eye movements

    PubMed Central

    Ono, Seiji

    2014-01-01

    Smooth pursuit eye movements allow us to maintain the image of a moving target on the fovea. Smooth pursuit consists of separate phases such as initiation and steady-state. These two phases are supported by different visual-motor mechanisms in cortical areas including the middle temporal (MT), the medial superior temporal (MST) cortex and the frontal eye field (FEF). Retinal motion signals are responsible for beginning the process of pursuit initiation, whereas extraretinal signals play a role in maintaining tracking speed. Smooth pursuit often requires on-line gain adjustments during tracking in response to a sudden change in target motion. For example, a brief sinusoidal perturbation of target motion induces a corresponding perturbation of eye motion. Interestingly, the perturbation ocular response is enhanced when baseline pursuit velocity is higher, even though the stimulus frequency and amplitude are constant. This on-line gain control mechanism is not simply due to visually driven activity of cortical neurons. Visual and pursuit signals are primarily processed in cortical MT/MST and the magnitude of perturbation responses could be regulated by the internal gain parameter in FEF. Furthermore, the magnitude and the gain slope of perturbation responses are altered by smooth pursuit adaptation using repeated trials of a step-ramp tracking with two different velocities (double-velocity paradigm). Therefore, smooth pursuit adaptation, which is attributed to the cerebellar plasticity mechanism, could affect the on-line gain control mechanism. PMID:24995378

  9. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  10. Multiscale Embedded Gene Co-expression Network Analysis

    PubMed Central

    Song, Won-Min; Zhang, Bin

    2015-01-01

    Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma. PMID:26618778

  11. Development of ion channels and neurofilaments during neuronal differentiation of mouse embryonal carcinoma cell lines.

    PubMed Central

    Kubo, Y

    1989-01-01

    1. an embryonal carcinoma cell line, PCC4-Aza1-ECA2, was induced to differentiate to neurones by two different procedures: an addition of retinoic acid to the culture medium or a reduction of serum concentration. The changes in membrane currents during differentiation were studied by the whole-cell variation of the patch-clamp technique and the change in neurofilament expression was studied immunohistochemically. 2. Stem cells showed the outward K+ current which inactivated slightly, but no inward currents were observed. These cells did not express neurofilament. 3. Three days after an addition of 10(-7) M-retinoic acid, neurofilament-positive round cells without processes began to appear. The inward currents observed in these cells were the Na+ current and fast-inactivating Ca2+-channel current. Four days after an addition of 10(-7) M-retinoic acid, the cells began to extend processes and showed an intense neurofilament expression. The inward currents were the Na+ current and slow-inactivating Ca2+-channel current, while the fast-inactivating Ca2+-channel current observed previously had almost disappeared. The amplitude of the outward K+ current was larger than that in the stem cell and it did not show clear inactivation. 4. By reducing the serum concentration in the medium from 10 to 0.1%, cells with processes were observed after 6 days. They were neurofilament-positive and had the Na+ current, both fast- and slow-inactivating Ca2+-channel currents, and the outward K+ current which inactivated slightly. 5. The properties of these ionic currents observed in induced neurones were studied. The Na+ current was blocked by 0.1 microM-tetrodotoxin at any stage. The Na+ current was evoked by a depolarization pulse to a level above -40 mV with a maximum amplitude at around -10 mV. The fast-inactivating Ca2+-channel current was evoked by a depolarization to a level above -50 mV with a maximum amplitude at around -15 mV. It was resistant to 50 microM-Cd2+. The slow

  12. The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

    SciTech Connect

    Flaskos, J.; Harris, W.; Sachana, M.; Munoz, D.; Tack, J.; Hargreaves, A.J. . E-mail: alan.hargreaves@ntu.ac.uk

    2007-03-15

    Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowth of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.

  13. Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin

    PubMed Central

    Wichmann, Heidi; Vocke, Farina; Brinkhoff, Thorsten; Simon, Meinhard; Richter-Landsberg, Christiane

    2015-01-01

    The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3–0.5 µg/mL (1.4–2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca2+-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death. PMID:26633426

  14. Cytotoxic Effects of Tropodithietic Acid on Mammalian Clonal Cell Lines of Neuronal and Glial Origin.

    PubMed

    Wichmann, Heidi; Vocke, Farina; Brinkhoff, Thorsten; Simon, Meinhard; Richter-Landsberg, Christiane

    2015-12-01

    The marine metabolite tropodithietic acid (TDA), produced by several Roseobacter clade bacteria, is known for its broad antimicrobial activity. TDA is of interest not only as a probiotic in aquaculture, but also because it might be of use as an antibacterial agent in non-marine or non-aquatic environments, and thus the potentially cytotoxic influences on eukaryotic cells need to be evaluated. The present study was undertaken to investigate its effects on cells of the mammalian nervous system, i.e., neuronal N2a cells and OLN-93 cells as model systems for nerve cells and glia. The data show that in both cell lines TDA exerted morphological changes and cytotoxic effects at a concentration of 0.3-0.5 µg/mL (1.4-2.4 µM). Furthermore, TDA caused a breakdown of the mitochondrial membrane potential, the activation of extracellular signal-regulated kinases ERK1/2, and the induction of the small heat shock protein HSP32/HO-1, which is considered as a sensor of oxidative stress. The cytotoxic effects were accompanied by an increase in intracellular Ca(2+)-levels, the disturbance of the microtubule network, and the reorganization of the microfilament system. Hence, mammalian cells are a sensitive target for the action of TDA and react by the activation of a stress response resulting in cell death. PMID:26633426

  15. Gene Co-Expression Analysis Predicts Genetic Variants Associated with Drug Responsiveness in Lung Cancer

    PubMed Central

    Shroff, Sanaya; Zhang, Jie; Huang, Kun

    2016-01-01

    Responsiveness to drugs is an important concern in designing personalized treatment for cancer patients. Currently genetic markers are often used to guide targeted therapy. However, deeper understanding of the molecular basis for drug responses and discovery of new predictive biomarkers for drug sensitivity are much needed. In this paper, we present a workflow for identifying condition-specific gene co-expression networks associated with responses to the tyrosine kinase inhibitor, Erlotinib, in lung adenocarcinoma cell lines using data from the Cancer Cell Line Encyclopedia by combining network mining and statistical analysis. Particularly, we have identified multiple gene modules specifically co-expressed in the drug responsive cell lines but not in the unresponsive group. Interestingly, most of these modules are enriched on specific cytobands, suggesting potential copy number variation events on these loci. Our results therefore imply that there are multiple genetic loci with copy number variations associated with the Erlotinib responses. The existence of CNVs in these loci is also confirmed in lung cancer tissue samples using the TCGA data. Since these structural variations are inferred from functional genomics data, these CNVs are functional variations. These results suggest the condition specific gene co- expression network mining approach is an effective approach in predicting candidate biomarkers for drug responses. PMID:27570645

  16. Gene Co-Expression Analysis Predicts Genetic Variants Associated with Drug Responsiveness in Lung Cancer.

    PubMed

    Shroff, Sanaya; Zhang, Jie; Huang, Kun

    2016-01-01

    Responsiveness to drugs is an important concern in designing personalized treatment for cancer patients. Currently genetic markers are often used to guide targeted therapy. However, deeper understanding of the molecular basis for drug responses and discovery of new predictive biomarkers for drug sensitivity are much needed. In this paper, we present a workflow for identifying condition-specific gene co-expression networks associated with responses to the tyrosine kinase inhibitor, Erlotinib, in lung adenocarcinoma cell lines using data from the Cancer Cell Line Encyclopedia by combining network mining and statistical analysis. Particularly, we have identified multiple gene modules specifically co-expressed in the drug responsive cell lines but not in the unresponsive group. Interestingly, most of these modules are enriched on specific cytobands, suggesting potential copy number variation events on these loci. Our results therefore imply that there are multiple genetic loci with copy number variations associated with the Erlotinib responses. The existence of CNVs in these loci is also confirmed in lung cancer tissue samples using the TCGA data. Since these structural variations are inferred from functional genomics data, these CNVs are functional variations. These results suggest the condition specific gene co- expression network mining approach is an effective approach in predicting candidate biomarkers for drug responses. PMID:27570645

  17. Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line.

    PubMed

    Scholz, Diana; Pöltl, Dominik; Genewsky, Andreas; Weng, Matthias; Waldmann, Tanja; Schildknecht, Stefan; Leist, Marcel

    2011-12-01

    We characterized phenotype and function of a fetal human mesencephalic cell line (LUHMES, Lund human mesencephalic) as neuronal model system. Neurodevelopmental profiling of the proliferation stage (d0, day 0) of these conditionally-immortalized cells revealed neuronal features, expressed simultaneously with some early neuroblast and stem cell markers. An optimized 2-step differentiation procedure, triggered by shut-down of the myc transgene, resulted in uniformly post-mitotic neurons within 5 days (d5). This was associated with down-regulation of some precursor markers and further up-regulation of neuronal genes. Neurite network formation involved the outgrowth of 1-2, often > 500 μm long projections. They showed dynamic growth cone behavior, as evidenced by time-lapse imaging of stably GFP-over-expressing cells. Voltage-dependent sodium channels and spontaneous electrical activity of LUHMES continuously increased from d0 to d11, while levels of synaptic markers reached their maximum on d5. The developmental expression patterns of most genes and of the dopamine uptake- and release-machinery appeared to be intrinsically predetermined, as the differentiation proceeded similarly when external factors such as dibutyryl-cAMP and glial cell derived neurotrophic factor were omitted. Only tyrosine hydroxylase required the continuous presence of cAMP. In conclusion, LUHMES are a robust neuronal model with adaptable phenotype and high value for neurodevelopmental studies, disease modeling and neuropharmacology. PMID:21434924

  18. Electroencephalogram evidence for the activation of human mirror neuron system during the observation of intransitive shadow and line drawing actions☆

    PubMed Central

    Zhu, Huaping; Sun, Yaoru; Wang, Fang

    2013-01-01

    Previous studies have demonstrated that hand shadows may activate the motor cortex associated with the mirror neuron system in human brain. However, there is no evidence of activity of the human mirror neuron system during the observation of intransitive movements by shadows and line drawings of hands. This study examined the suppression of electroencephalography mu waves (8–13 Hz) induced by observation of stimuli in 18 healthy students. Three stimuli were used: real hand actions, hand shadow actions and actions made by line drawings of hands. The results showed significant desynchronization of the mu rhythm (“mu suppression”) across the sensorimotor cortex (recorded at C3, Cz and C4), the frontal cortex (recorded at F3, Fz and F4) and the central and right posterior parietal cortex (recorded at Pz and P4) under all three conditions. Our experimental findings suggest that the observation of “impoverished hand actions”, such as intransitive movements of shadows and line drawings of hands, is able to activate widespread cortical areas related to the putative human mirror neuron system. PMID:25206595

  19. Learning from Co-expression Networks: Possibilities and Challenges

    PubMed Central

    Serin, Elise A. R.; Nijveen, Harm; Hilhorst, Henk W. M.; Ligterink, Wilco

    2016-01-01

    Plants are fascinating and complex organisms. A comprehensive understanding of the organization, function and evolution of plant genes is essential to disentangle important biological processes and to advance crop engineering and breeding strategies. The ultimate aim in deciphering complex biological processes is the discovery of causal genes and regulatory mechanisms controlling these processes. The recent surge of omics data has opened the door to a system-wide understanding of the flow of biological information underlying complex traits. However, dealing with the corresponding large data sets represents a challenging endeavor that calls for the development of powerful bioinformatics methods. A popular approach is the construction and analysis of gene networks. Such networks are often used for genome-wide representation of the complex functional organization of biological systems. Network based on similarity in gene expression are called (gene) co-expression networks. One of the major application of gene co-expression networks is the functional annotation of unknown genes. Constructing co-expression networks is generally straightforward. In contrast, the resulting network of connected genes can become very complex, which limits its biological interpretation. Several strategies can be employed to enhance the interpretation of the networks. A strategy in coherence with the biological question addressed needs to be established to infer reliable networks. Additional benefits can be gained from network-based strategies using prior knowledge and data integration to further enhance the elucidation of gene regulatory relationships. As a result, biological networks provide many more applications beyond the simple visualization of co-expressed genes. In this study we review the different approaches for co-expression network inference in plants. We analyse integrative genomics strategies used in recent studies that successfully identified candidate genes taking advantage of

  20. Co-Expression of GRK2 Reveals a Novel Conformational State of the µ-Opioid Receptor

    PubMed Central

    Nickolls, Sarah A.; Humphreys, Sian; Clark, Mellissa; McMurray, Gordon

    2013-01-01

    Agonists at the µ-opioid receptor are known to produce potent analgesic responses in the clinical setting, therefore, an increased understanding of the molecular interactions of ligands at this receptor could lead to improved analgesics. As historically morphine has been shown to be a poor recruiter of β-arrestin in recombinant cell systems and this can be overcome by the co-expression of GRK2, we investigated the effects of GRK2 co-expression, in a recombinant µ-opioid receptor cell line, on ligand affinity and intrinsic activity in both β-arrestin recruitment and [35S]GTPγS binding assays. We also investigated the effect of receptor depletion in the β-arrestin assay. GRK2 co-expression increased both agonist Emax and potency in the β-arrestin assay. The increase in agonist potency could not be reversed using receptor depletion, supporting that the effects were due to a novel receptor conformation not system amplification. We also observed a small but significant effect on agonist KL values. Potency values in the [35S]GTPγS assay were unchanged; however, inverse agonist activity became evident with GRK2 co-expression. We conclude that this is direct evidence that the µ-opioid receptor is an allosteric protein and the co-expression of signalling molecules elicits changes in its conformation and thus ligand affinity. This has implications when describing how ligands interact with the receptor and how efficacy is determined. PMID:24376730

  1. Effects of low frequency electric fields on synaptic integration in hippocampal CA1 pyramidal neurons: implications for power line emissions

    PubMed Central

    Cavarretta, Francesco; Carnevale, Nicholas T.; Tegolo, Domenico; Migliore, Michele

    2014-01-01

    The possible cognitive effects of low frequency external electric fields (EFs), such as those generated by power lines, are poorly understood. Their functional consequences for mechanisms at the single neuron level are very difficult to study and identify experimentally, especially in vivo. The major open problem is that experimental investigations on humans have given inconsistent or contradictory results, making it difficult to estimate the possible effects of external low frequency electric fields on cognitive functions. Here we investigate this issue with realistic models of hippocampal CA1 pyramidal neurons. Our findings suggest how and why EFs, with environmentally observed frequencies and intensities far lower than what is required for direct neural activation, can perturb dendritic signal processing and somatic firing of neurons that are crucially involved in cognitive tasks such as learning and memory. These results show that individual neuronal morphology, ion channel dendritic distribution, and alignment with the electric field are major determinants of overall effects, and provide a physiologically plausible explanation of why experimental findings can appear to be small and difficult to reproduce, yet deserve serious consideration. PMID:25346660

  2. Shared Pathways Among Autism Candidate Genes Determined by Co-expression Network Analysis of the Developing Human Brain Transcriptome.

    PubMed

    Mahfouz, Ahmed; Ziats, Mark N; Rennert, Owen M; Lelieveldt, Boudewijn P F; Reinders, Marcel J T

    2015-12-01

    Autism spectrum disorder (ASD) is a neurodevelopmental syndrome known to have a significant but complex genetic etiology. Hundreds of diverse genes have been implicated in ASD; yet understanding how many genes, each with disparate function, can all be linked to a single clinical phenotype remains unclear. We hypothesized that understanding functional relationships between autism candidate genes during normal human brain development may provide convergent mechanistic insight into the genetic heterogeneity of ASD. We analyzed the co-expression relationships of 455 genes previously implicated in autism using the BrainSpan human transcriptome database, across 16 anatomical brain regions spanning prenatal life through adulthood. We discovered modules of ASD candidate genes with biologically relevant temporal co-expression dynamics, which were enriched for functional ontologies related to synaptogenesis, apoptosis, and GABA-ergic neurons. Furthermore, we also constructed co-expression networks from the entire transcriptome and found that ASD candidate genes were enriched in modules related to mitochondrial function, protein translation, and ubiquitination. Hub genes central to these ASD-enriched modules were further identified, and their functions supported these ontological findings. Overall, our multi-dimensional co-expression analysis of ASD candidate genes in the normal developing human brain suggests the heterogeneous set of ASD candidates share transcriptional networks related to synapse formation and elimination, protein turnover, and mitochondrial function. PMID:26399424

  3. A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

    NASA Astrophysics Data System (ADS)

    Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

    1989-06-01

    The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

  4. The novel neuropeptide phoenixin is highly co-expressed with nesfatin-1 in the rat hypothalamus, an immunohistochemical study.

    PubMed

    Pałasz, Artur; Rojczyk, Ewa; Bogus, Katarzyna; Worthington, John J; Wiaderkiewicz, Ryszard

    2015-04-10

    The hypothalamus regulates a number of autonomic functions essential for homeostasis; therefore, investigations concerning hypothalamic neuropeptides and their functions and distribution are of great importance in contemporary neuroscience. Recently, novel regulatory factors expressed in the hypothalamus have been discovered, of which nesfatin-1 and phoenixin (PNX), show intriguing similarities in their brain distributions. There are currently few studies characterizing PNX expression, so it is imperative to accurately trace its localization, with particular attention to the hypothalamic nuclei and nesfatin-1 co-expression. Using fluorescence and classical immunohistochemical stainings on adult rat brain, we visualized the potential co-expression of nesfatin-1 and PNX immunoreactive cells. We have demonstrated a distinct PNX-immunoreactivity in 21-32% of cells in the arcuate nucleus, paraventricular nucleus, ventromedial and lateral hypothalamus. Nesfatin-1 expression reached 45-68% of all neurons in the same sites, while co-expression was strikingly seen in the vast majority (70-86%) of PNX-immunoreactive neurons in the rat hypothalamic nuclei. Our results demonstrate for the first time, a wide distribution of PNX in the hypothalamus which could implicate a potential functional relationship with nesfatin-1, possibly in the regulation of the hypothalamic-pituitary-gonadal axis or other autonomic functions, which require further study. PMID:25736948

  5. Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line

    SciTech Connect

    Blusztajn, J.K.; Liscovitch, M.; Richardson, U.I.

    1987-08-01

    Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used a precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here the authors used a population of purely cholinergic cells (human neuroblastomas, LA-N-2), incubated in the presence of (methyl-/sup 3/H)methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. Three peaks of radioactive material that cochromatographed with authentic AcCho, choline, and phosphocholine were observed when the water-soluble metabolites of the (/sup 3/H)PtdCho were purified by high-performance liquid chromatography. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine.

  6. Electrophysiology Alterations in Primary Visual Cortex Neurons of Retinal Degeneration (S334ter-line-3) Rats

    PubMed Central

    Chen, Ke; Wang, Yi; Liang, Xiaohua; Zhang, Yihuai; Ng, Tsz Kin; Chan, Leanne Lai Hang

    2016-01-01

    The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration. PMID:27225415

  7. Electrophysiology Alterations in Primary Visual Cortex Neurons of Retinal Degeneration (S334ter-line-3) Rats.

    PubMed

    Chen, Ke; Wang, Yi; Liang, Xiaohua; Zhang, Yihuai; Ng, Tsz Kin; Chan, Leanne Lai Hang

    2016-01-01

    The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration. PMID:27225415

  8. Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis

    PubMed Central

    Creanza, Teresa Maria; Liguori, Maria; Liuni, Sabino; Nuzziello, Nicoletta; Ancona, Nicola

    2016-01-01

    Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment. PMID:27314336

  9. Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis.

    PubMed

    Creanza, Teresa Maria; Liguori, Maria; Liuni, Sabino; Nuzziello, Nicoletta; Ancona, Nicola

    2016-01-01

    Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment. PMID:27314336

  10. A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro.

    PubMed

    Coleman, Michael D; O'Neil, John D; Woehrling, Elizabeth K; Ndunge, Oscar Bate Akide; Hill, Eric J; Menache, Andre; Reiss, Claude J

    2012-01-01

    Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health. PMID:22880100

  11. A Preliminary Investigation into the Impact of a Pesticide Combination on Human Neuronal and Glial Cell Lines In Vitro

    PubMed Central

    Coleman, Michael D.; O'Neil, John D.; Woehrling, Elizabeth K.; Ndunge, Oscar Bate Akide; Hill, Eric J.; Menache, Andre; Reiss, Claude J.

    2012-01-01

    Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health. PMID:22880100

  12. Dynamic Visualization of Co-expression in Systems Genetics Data

    SciTech Connect

    New, Joshua Ryan; Huang, Jian; Chesler, Elissa J

    2008-01-01

    Biologists hope to address grand scientific challenges by exploring the abundance of data made available through modern microarray technology and other high-throughput techniques. The impact of this data, however, is limited unless researchers can effectively assimilate such complex information and integrate it into their daily research; interactive visualization tools are called for to support the effort. Specifically, typical studies of gene co-expression require novel visualization tools that enable the dynamic formulation and fine-tuning of hypotheses to aid the process of evaluating sensitivity of key parameters. These tools should allow biologists to develop an intuitive understanding of the structure of biological networks and discover genes which reside in critical positions in networks and pathways. By using a graph as a universal data representation of correlation in gene expression data, our novel visualization tool employs several techniques that when used in an integrated manner provide innovative analytical capabilities. Our tool for interacting with gene co-expression data integrates techniques such as: graph layout, qualitative subgraph extraction through a novel 2D user interface, quantitative subgraph extraction using graph-theoretic algorithms or by querying an optimized b-tree, dynamic level-of-detail graph abstraction, and template-based fuzzy classification using neural networks. We demonstrate our system using a real-world workflow from a large-scale, systems genetics study of mammalian gene co-expression.

  13. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  14. Agmatine induces Nrf2 and protects against corticosterone effects in hippocampal neuronal cell line.

    PubMed

    Freitas, Andiara E; Egea, Javier; Buendía, Izaskun; Navarro, Elisa; Rada, Patricia; Cuadrado, Antonio; Rodrigues, Ana Lúcia S; López, Manuela G

    2015-01-01

    Hyperactivation of the hypothalamic-pituitary-adrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration- and time-dependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α2-adrenoceptor antagonist), ketanserin (5-HT2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α2-adrenergic and 5-HT2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression. PMID:25084759

  15. Arabidopsis gene co-expression network and its functional modules

    PubMed Central

    Mao, Linyong; Van Hemert, John L; Dash, Sudhansu; Dickerson, Julie A

    2009-01-01

    Background Biological networks characterize the interactions of biomolecules at a systems-level. One important property of biological networks is the modular structure, in which nodes are densely connected with each other, but between which there are only sparse connections. In this report, we attempted to find the relationship between the network topology and formation of modular structure by comparing gene co-expression networks with random networks. The organization of gene functional modules was also investigated. Results We constructed a genome-wide Arabidopsis gene co-expression network (AGCN) by using 1094 microarrays. We then analyzed the topological properties of AGCN and partitioned the network into modules by using an efficient graph clustering algorithm. In the AGCN, 382 hub genes formed a clique, and they were densely connected only to a small subset of the network. At the module level, the network clustering results provide a systems-level understanding of the gene modules that coordinate multiple biological processes to carry out specific biological functions. For instance, the photosynthesis module in AGCN involves a very large number (> 1000) of genes which participate in various biological processes including photosynthesis, electron transport, pigment metabolism, chloroplast organization and biogenesis, cofactor metabolism, protein biosynthesis, and vitamin metabolism. The cell cycle module orchestrated the coordinated expression of hundreds of genes involved in cell cycle, DNA metabolism, and cytoskeleton organization and biogenesis. We also compared the AGCN constructed in this study with a graphical Gaussian model (GGM) based Arabidopsis gene network. The photosynthesis, protein biosynthesis, and cell cycle modules identified from the GGM network had much smaller module sizes compared with the modules found in the AGCN, respectively. Conclusion This study reveals new insight into the topological properties of biological networks. The

  16. Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in enhanced green fluorescent protein-expressing transgenic medaka lines.

    PubMed

    Takahashi, Akiko; Islam, M Sadiqul; Abe, Hideki; Okubo, Kataaki; Akazome, Yasuhisa; Kaneko, Takeshi; Hioki, Hiroyuki; Oka, Yoshitaka

    2016-03-01

    Teleosts possess two or three paralogs of gonadotropin-releasing hormone (GnRH) genes: gnrh1, gnrh2, and gnrh3. Some species have lost the gnrh1 and/or gnrh3 genes, whereas gnrh2 has been completely conserved in the teleost species analyzed to date. In most teleosts that possess gnrh1, GnRH1 peptide is the authentic GnRH that stimulates gonadotropin release, whereas GnRH2 and GnRH3, if present, are neuromodulatory. Progenitors of GnRH1 and GnRH3 neurons originate from olfactory placodes and migrate to their destination during early development. However, because of the relatively low affinity/specificity of generally available antibodies that recognize GnRH1 or GnRH3, labeling of these neurons has only been possible using genetic manipulation. We used a model teleost, medaka, which possesses all three paralogous gnrh genes, to analyze development of forebrain GnRH neurons composed of GnRH1 and GnRH3 neurons. Here, we newly generated transgenic medaka lines that express enhanced green fluorescent protein under the control of promoters for gnrh1 or gnrh3, to detect GnRH neurons and facilitate immunohistochemical analysis of the neuronal morphology. We used a combination of immunohistochemistry and three-dimensional confocal microscopy image reconstructions to improve identification of neurites from GnRH1 or GnRH3 neuronal populations with greater precision. This led us to clearly identify the hypophysiotropic innervation of GnRH1 neurons residing in the ventral preoptic area (vPOA) from as early as 10 days post hatching. Furthermore, these analyses also revealed retinopetal projections of nonhypophysiotropic GnRH1 neurons in vPOA, prominent during early developmental stages, and multiple populations of GnRH3 neurons with different origins and migratory pathways. PMID:26287569

  17. Gene co-expression networks shed light into diseases of brain iron accumulation

    PubMed Central

    Bettencourt, Conceição; Forabosco, Paola; Wiethoff, Sarah; Heidari, Moones; Johnstone, Daniel M.; Botía, Juan A.; Collingwood, Joanna F.; Hardy, John; Milward, Elizabeth A.; Ryten, Mina; Houlden, Henry

    2016-01-01

    Aberrant brain iron deposition is observed in both common and rare neurodegenerative disorders, including those categorized as Neurodegeneration with Brain Iron Accumulation (NBIA), which are characterized by focal iron accumulation in the basal ganglia. Two NBIA genes are directly involved in iron metabolism, but whether other NBIA-related genes also regulate iron homeostasis in the human brain, and whether aberrant iron deposition contributes to neurodegenerative processes remains largely unknown. This study aims to expand our understanding of these iron overload diseases and identify relationships between known NBIA genes and their main interacting partners by using a systems biology approach. We used whole-transcriptome gene expression data from human brain samples originating from 101 neuropathologically normal individuals (10 brain regions) to generate weighted gene co-expression networks and cluster the 10 known NBIA genes in an unsupervised manner. We investigated NBIA-enriched networks for relevant cell types and pathways, and whether they are disrupted by iron loading in NBIA diseased tissue and in an in vivo mouse model. We identified two basal ganglia gene co-expression modules significantly enriched for NBIA genes, which resemble neuronal and oligodendrocytic signatures. These NBIA gene networks are enriched for iron-related genes, and implicate synapse and lipid metabolism related pathways. Our data also indicates that these networks are disrupted by excessive brain iron loading. We identified multiple cell types in the origin of NBIA disorders. We also found unforeseen links between NBIA networks and iron-related processes, and demonstrate convergent pathways connecting NBIAs and phenotypically overlapping diseases. Our results are of further relevance for these diseases by providing candidates for new causative genes and possible points for therapeutic intervention. PMID:26707700

  18. Gene co-expression networks shed light into diseases of brain iron accumulation.

    PubMed

    Bettencourt, Conceição; Forabosco, Paola; Wiethoff, Sarah; Heidari, Moones; Johnstone, Daniel M; Botía, Juan A; Collingwood, Joanna F; Hardy, John; Milward, Elizabeth A; Ryten, Mina; Houlden, Henry

    2016-03-01

    Aberrant brain iron deposition is observed in both common and rare neurodegenerative disorders, including those categorized as Neurodegeneration with Brain Iron Accumulation (NBIA), which are characterized by focal iron accumulation in the basal ganglia. Two NBIA genes are directly involved in iron metabolism, but whether other NBIA-related genes also regulate iron homeostasis in the human brain, and whether aberrant iron deposition contributes to neurodegenerative processes remains largely unknown. This study aims to expand our understanding of these iron overload diseases and identify relationships between known NBIA genes and their main interacting partners by using a systems biology approach. We used whole-transcriptome gene expression data from human brain samples originating from 101 neuropathologically normal individuals (10 brain regions) to generate weighted gene co-expression networks and cluster the 10 known NBIA genes in an unsupervised manner. We investigated NBIA-enriched networks for relevant cell types and pathways, and whether they are disrupted by iron loading in NBIA diseased tissue and in an in vivo mouse model. We identified two basal ganglia gene co-expression modules significantly enriched for NBIA genes, which resemble neuronal and oligodendrocytic signatures. These NBIA gene networks are enriched for iron-related genes, and implicate synapse and lipid metabolism related pathways. Our data also indicates that these networks are disrupted by excessive brain iron loading. We identified multiple cell types in the origin of NBIA disorders. We also found unforeseen links between NBIA networks and iron-related processes, and demonstrate convergent pathways connecting NBIAs and phenotypically overlapping diseases. Our results are of further relevance for these diseases by providing candidates for new causative genes and possible points for therapeutic intervention. PMID:26707700

  19. An MMIC implementation of FitzHugh-Nagumo neurons using a resonant tunneling diode nonlinear transmission line

    NASA Astrophysics Data System (ADS)

    Klofaï, Yerima; Essimbi, B. Z.; Jäger, D.

    2015-02-01

    In this paper the electronic implementation of FitzHugh-Nagumo (F-N) neurons via monolithic microwave integrated circuits (MMIC) based upon a resonant tunneling diode (RTD) nonlinear transmission line (NLTL) using a coplanar waveguide (CPW) is considered. The goals are twofold. In the framework of electrical equivalent circuit emulating nonlinear active wave propagation effects, it is shown, on one hand, how different physical mechanisms are responsible for the time evolution of given input signals. A key result is that this medium supports stable and stationary pulse propagation that is only determined by the parameters of the RTD-NLTL and is independent of the boundary conditions. On the other hand, the influence of specific line elements on the output signal waveform is discussed in a most systematic manner. This leads, for the first time, to a more physical interpretation of the properties of the RTD-NLTL and, furthermore, to interesting technical applications at multi-GHz frequencies and on picosecond time scales. As a result, physically based ways are elucidated regarding how the technical design of those compact neuromorphic electrical circuits can be optimized by numerical simulations and performed using standard MMIC technologies.

  20. A sensory labeled-line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia

    PubMed Central

    Knowlton, Wendy M.; Palkar, Radhika; Lippoldt, Erika K.; McCoy, Daniel D.; Baluch, Farhan; Chen, Jessica; McKemy, David D.

    2013-01-01

    Many primary sensory neurons are polymodal, responding to multiple stimulus modalities (chemical, thermal, or mechanical), yet each modality is recognized differently. While polymodality implies that stimulus encoding occurs in higher centers such as the spinal cord or brain, recent sensory neuron ablation studies find that behavioral responses to different modalities require distinct subpopulations, suggesting the existence of modality-specific labeled-lines at the level of the sensory afferent. Here we provide evidence that neurons expressing TRPM8, a cold- and menthol-gated channel required for normal cold responses in mammals, represents a labeled-line solely for cold sensation. We examined the behavioral significance of conditionally ablating TRPM8+ neurons in adult mice, finding that, like animals lacking TRPM8 channels (Trpm8−/−), animals depleted of TRPM8 neurons (ablated) are insensitive to cool to painfully cold temperatures. Ablated animals showed little aversion to noxious cold and did not distinguish between cold and a preferred warm temperature, a phenotype more profound than that of Trpm8−/− mice which exhibit only partial cold avoidance and preference behaviors. In addition to acute responses, cold pain associated with inflammation and nerve injury was significantly attenuated in ablated and Trpm8−/− mice. Moreover, cooling-induced analgesia after nerve injury was abolished in both genotypes. Lastly, heat, mechanical, and proprioceptive behaviors were normal in ablated mice, demonstrating that TRPM8 neurons are dispensable for other somatosensory modalities. Together these data show that while some limited cold sensitivity remains in Trpm8−/− mice, TRPM8 neurons are required for the breadth of behavioral responses evoked by cold temperatures. PMID:23407943

  1. Transcription factor-pathway co-expression analysis reveals cooperation between SP1 and ESR1 on dysregulating cell cycle arrest in non-hyperdiploid multiple myeloma

    PubMed Central

    Wang, Xujun; Yan, Zhenyu; Fulciniti, Mariateresa; Li, Yingxiang; Gkotzamanidou, Maria; Amin, Samir B; Shah, Parantu K; Zhang, Yong

    2014-01-01

    Multiple myeloma is a hematological cancer of plasma B-cells and remains incurable. Two major subtypes of myeloma, hyperdiploid (HMM) and non-hyperdiploid myeloma (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis but their dysregulation in myeloma subtypes are less studied. Here we develop a TrF-pathway co-expression analysis to identify altered co-expression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, NF-κB and HOXA9 have significantly lower co-expression with cell cycle arrest in HMM, co-occurring with their over-activation in HMM. In contrast, TrFs ESR1, SP1 and E2F1 have significantly lower co-expression with cell cycle arrest in NHMM. SP1 ChIP targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their over-activation in NHMM disrupts proper regulation of cell cycle arrest. Co-targeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway co-expression dysregulation in human cancers facilitates forming novel hypotheses towards clinical utility. PMID:23925045

  2. Differential Co-Expression between α-Synuclein and IFN-γ Signaling Genes across Development and in Parkinson’s Disease

    PubMed Central

    Liscovitch, Noa; French, Leon

    2014-01-01

    Expression patterns of the alpha-synuclein gene (SNCA) were studied across anatomy, development, and disease to better characterize its role in the brain. In this postmortem study, negative spatial co-expression between SNCA and 73 interferon-γ (IFN-γ) signaling genes was observed across many brain regions. Recent animal studies have demonstrated that IFN-γ induces loss of dopamine neurons and nigrostriatal degeneration. This opposing pattern between SNCA and IFN-γ signaling genes increases with age (rho = −0.78). In contrast, a meta-analysis of four microarray experiments representing 126 substantia nigra samples reveals a switch to positive co-expression in Parkinson’s disease (p<0.005). Use of genome-wide testing demonstrates this relationship is specific to SNCA (p<0.002). This change in co-expression suggests an immunomodulatory role of SNCA that may provide insight into neurodegeneration. Genes showing similar co-expression patterns have been previously linked to Alzheimer’s (ANK1) and Parkinson’s disease (UBE2E2, PCMT1, HPRT1 and RIT2). PMID:25493648

  3. N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson's Disease: Preliminary Clinical and Cell Line Data

    PubMed Central

    Monti, Daniel A.; Zabrecky, George; Kremens, Daniel; Liang, Tsao-Wei; Wintering, Nancy A.; Cai, Jingli; Wei, Xiatao; Bazzan, Anthony J.; Zhong, Li; Bowen, Brendan; Intenzo, Charles M.; Iacovitti, Lorraine; Newberg, Andrew B.

    2016-01-01

    Backgound The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson’s disease (PD). Methods The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson’s Disease Rating Scale (UPDRS) to measure clinical symptoms. Results The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p<0.05 for all values) in the PD group treated with NAC, and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%, p = 0.01). Conclusions The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. Trial Registration ClinicalTrials.gov NCT02445651

  4. Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death.

    PubMed

    Hemendinger, Richelle A; Armstrong, Edward J; Brooks, Benjamin Rix

    2011-03-15

    Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC₅₀ (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC₅₀ (concentration at which 50% of maximal cell death is inhibited) of 0.6 μM and 0.4 μM, respectively. In contrast, MTHF (up to 10 μM) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS. PMID:21237187

  5. Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

    SciTech Connect

    Hemendinger, Richelle A. Armstrong, Edward J.; Brooks, Benjamin Rix

    2011-03-15

    Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC{sub 50} (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC{sub 50} (concentration at which 50% of maximal cell death is inhibited) of 0.6 {mu}M and 0.4 {mu}M, respectively. In contrast, MTHF (up to 10 {mu}M) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.

  6. KL/KIT co-expression in mouse fetal oocytes.

    PubMed

    Doneda, Luisa; Klinger, Francesca-Gioia; Larizza, Lidia; De Felici, Massimo

    2002-12-01

    The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL. PMID:12533025

  7. Co-expression of glutaminase K and L isoenzymes in human tumour cells

    PubMed Central

    2004-01-01

    The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states. PMID:15496140

  8. Characterization of Tusc5, a Unique Adipocyte Gene Co-Expressed in Peripheral Somatosensory Neurons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tumor suppressor candidate 5 (Tusc5, GenBank nomenclature) is a cold-repressed gene encoding a member of the CD225 domain-containing family, identified through analysis of transcripts differentially-expressed in brown adipose tissue (BAT) with changes in ambient temperature. Tusc5 mRNA was found to ...

  9. Visualizing estrogen receptor-a-expressing neurons using a new ERa-ZsGreen reporter mouse line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A variety of biological functions of estrogens, including regulation of energy metabolism, are mediated by neurons expressingestrogen receptor-a (ERa) in the brain. However, complex intracellular processes in these ERa-expressing neurons are difficult to unravel, due to the lack of strategy to visua...

  10. Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

    PubMed

    Tanaka, Misaki; Fujiwara, Ai; Suzuki, Akio; Yamasaki, Takeshi; Hasebe, Rie; Masujin, Kentaro; Horiuchi, Motohiro

    2016-08-01

    We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation. PMID:27267758

  11. Co-Expression of Cancer Stem Cell Markers Corresponds to a Pro-Tumorigenic Expression Profile in Pancreatic Adenocarcinoma.

    PubMed

    Skoda, Jan; Hermanova, Marketa; Loja, Tomas; Nemec, Pavel; Neradil, Jakub; Karasek, Petr; Veselska, Renata

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers-CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin-by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile. PMID:27414409

  12. Co-Expression of Cancer Stem Cell Markers Corresponds to a Pro-Tumorigenic Expression Profile in Pancreatic Adenocarcinoma

    PubMed Central

    Skoda, Jan; Hermanova, Marketa; Loja, Tomas; Nemec, Pavel; Neradil, Jakub; Karasek, Petr; Veselska, Renata

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Its dismal prognosis is often attributed to the presence of cancer stem cells (CSCs) that have been identified in PDAC using various markers. However, the co-expression of all of these markers has not yet been evaluated. Furthermore, studies that compare the expression levels of CSC markers in PDAC tumor samples and in cell lines derived directly from those tumors are lacking. Here, we analyzed the expression of putative CSC markers—CD24, CD44, epithelial cell adhesion molecule (EpCAM), CD133, and nestin—by immunofluorescence, flow cytometry and quantitative PCR in 3 PDAC-derived cell lines and by immunohistochemistry in 3 corresponding tumor samples. We showed high expression of the examined CSC markers among all of the cell lines and tumor samples, with the exception of CD24 and CD44, which were enriched under in vitro conditions compared with tumor tissues. The proportions of cells positive for the remaining markers were comparable to those detected in the corresponding tumors. Co-expression analysis using flow cytometry revealed that CD24+/CD44+/EpCAM+/CD133+ cells represented a significant population of the cells (range, 43 to 72%) among the cell lines. The highest proportion of CD24+/CD44+/EpCAM+/CD133+ cells was detected in the cell line derived from the tumor of a patient with the shortest survival. Using gene expression profiling, we further identified the specific pro-tumorigenic expression profile of this cell line compared with the profiles of the other two cell lines. Together, CD24+/CD44+/EpCAM+/CD133+ cells are present in PDAC cell lines derived from primary tumors, and their increased proportion corresponds with a pro-tumorigenic gene expression profile. PMID:27414409

  13. Investigating the Combinatory Effects of Biological Networks on Gene Co-expression

    PubMed Central

    Zhang, Cheng; Lee, Sunjae; Mardinoglu, Adil; Hua, Qiang

    2016-01-01

    Co-expressed genes often share similar functions, and gene co-expression networks have been widely used in studying the functionality of gene modules. Previous analysis indicated that genes are more likely to be co-expressed if they are either regulated by the same transcription factors, forming protein complexes or sharing similar topological properties in protein-protein interaction networks. Here, we reconstructed transcriptional regulatory and protein-protein networks for Saccharomyces cerevisiae using well-established databases, and we evaluated their co-expression activities using publically available gene expression data. Based on our network-dependent analysis, we found that genes that were co-regulated in the transcription regulatory networks and shared similar neighbors in the protein-protein networks were more likely to be co-expressed. Moreover, their biological functions were closely related. PMID:27445830

  14. Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3.

    PubMed

    Jalalvand, Elham; Robertson, Brita; Wallén, Peter; Grillner, Sten

    2016-01-01

    Cerebrospinal fluid-contacting (CSF-c) cells are found in all vertebrates but their function has remained elusive. We recently identified one type of laterally projecting CSF-c cell in lamprey spinal cord with neuronal properties that expresses GABA and somatostatin. We show here that these CSF-c neurons respond to both mechanical stimulation and to lowered pH. These effects are most likely mediated by ASIC3-channels, since APETx2, a specific antagonist of ASIC3, blocks them both. Furthermore, lowering of pH as well as application of somatostatin will reduce the locomotor burst rate. The somatostatin receptor antagonist counteracts the effects of both a decrease in pH and of somatostatin. Lateral bending movement imposed on the spinal cord, as would occur during natural swimming, activates CSF-c neurons. Taken together, we show that CSF-c neurons act both as mechanoreceptors and as chemoreceptors through ASIC3 channels, and their action may protect against pH-changes resulting from excessive neuronal activity. PMID:26743691

  15. Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

    PubMed Central

    Jalalvand, Elham; Robertson, Brita; Wallén, Peter; Grillner, Sten

    2016-01-01

    Cerebrospinal fluid-contacting (CSF-c) cells are found in all vertebrates but their function has remained elusive. We recently identified one type of laterally projecting CSF-c cell in lamprey spinal cord with neuronal properties that expresses GABA and somatostatin. We show here that these CSF-c neurons respond to both mechanical stimulation and to lowered pH. These effects are most likely mediated by ASIC3-channels, since APETx2, a specific antagonist of ASIC3, blocks them both. Furthermore, lowering of pH as well as application of somatostatin will reduce the locomotor burst rate. The somatostatin receptor antagonist counteracts the effects of both a decrease in pH and of somatostatin. Lateral bending movement imposed on the spinal cord, as would occur during natural swimming, activates CSF-c neurons. Taken together, we show that CSF-c neurons act both as mechanoreceptors and as chemoreceptors through ASIC3 channels, and their action may protect against pH-changes resulting from excessive neuronal activity. PMID:26743691

  16. Effects of Oxidative Stress and Testosterone on Pro-Inflammatory Signaling in a Female Rat Dopaminergic Neuronal Cell Line.

    PubMed

    Holmes, Shaletha; Singh, Meharvan; Su, Chang; Cunningham, Rebecca L

    2016-07-01

    Parkinson's disease, a progressive neurodegenerative disorder, is associated with oxidative stress and neuroinflammation. These pathological markers can contribute to the loss of dopamine neurons in the midbrain. Interestingly, men have a 2-fold increased incidence for Parkinson's disease than women. Although the mechanisms underlying this sex difference remain elusive, we propose that the primary male sex hormone, testosterone, is involved. Our previous studies show that testosterone, through a putative membrane androgen receptor, can increase oxidative stress-induced neurotoxicity in dopamine neurons. Based on these results, this study examines the role of nuclear factor κ B (NF-κB), cyclooxygenase-2 (COX2), and apoptosis in the deleterious effects of androgens in an oxidative stress environment. We hypothesize, under oxidative stress environment, testosterone via a putative membrane androgen receptor will exacerbate oxidative stress-induced NF-κB/COX2 signaling in N27 dopaminergic neurons, leading to apoptosis. Our data show that testosterone increased the expression of COX2 and apoptosis in dopamine neurons. Inhibiting the NF-κB and COX2 pathway with CAPE and ibuprofen, respectively, blocked testosterone's negative effects on cell viability, indicating that NF-κB/COX2 cascade plays a role in the negative interaction between testosterone and oxidative stress on neuroinflammation. These data further support the role of testosterone mediating the loss of dopamine neurons under oxidative stress conditions, which may be a key mechanism contributing to the increased incidence of Parkinson's disease in men compared with women. PMID:27167771

  17. HLXB9 Gene Expression, and Nuclear Location during In Vitro Neuronal Differentiation in the SK-N-BE Neuroblastoma Cell Line

    PubMed Central

    Leotta, Claudia Giovanna; Federico, Concetta; Brundo, Maria Violetta; Tosi, Sabrina; Saccone, Salvatore

    2014-01-01

    Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways. PMID:25136833

  18. Transcriptional activation of JC virus by human T-lymphotropic virus type I Tax protein in human neuronal cell lines.

    PubMed

    Okada, Y; Sawa, H; Tanaka, S; Takada, A; Suzuki, S; Hasegawa, H; Umemura, T; Fujisawa, J; Tanaka, Y; Hall, W W; Nagashima, K

    2000-06-01

    Polyomavirus JC (JCV) causes the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). The recent demonstration of cases of PML in association with human T-lymphotropic virus type I (HTLV-I) infection prompted us to examine whether the HTLV-I-encoded regulatory protein Tax activates JCV transcription. By employing a dual luciferase assay, we initially found that the expression of Tax activated the transcriptional potential of both early and late promoters of JCV in human neuronal but not in non-neuronal cells. We subsequently analyzed the mechanism of Tax-induced activation of the JCV promoter in neuronal cells with the following results: 1) the JCV promoter that lacks the NF-kappaB-binding motif could not be activated by Tax; 2) the overexpression of IkappaBalpha abolished Tax-induced transcriptional activation of the JCV promoter; 3) a Tax mutant (M22) lacking the potential for activation via the NF-kappaB pathway did not activate the JCV promoter. Furthermore, Tax enhances the gene expression of JCV T antigen and VP1. We examined mechanisms of the cell-specific activation of the JCV promoter by Tax. Electrophoretic mobility shift assay demonstrated the presence of Tax-bound protein(s) that were specifically present in non-neuronal cells. This study is the first demonstration of the activation of JCV promoter by HTLV-I Tax in an NF-kappaB-dependent manner. PMID:10828075

  19. Interaction and colocalization of HERMES/RBPMS with NonO, PSF, and G3BP1 in neuronal cytoplasmic RNP granules in mouse retinal line cells.

    PubMed

    Furukawa, Mari T; Sakamoto, Hiroshi; Inoue, Kunio

    2015-04-01

    HERMES, also called RBPMS, is a conserved RNA binding protein with a single RNA recognition motif (RRM) that is abundantly expressed in retinal ganglion cells (RGCs) and in the heart in vertebrates. Here, we identified NonO and PSF as the interacting proteins of HERMES only when the neuronal differentiation of the retinal cell line RGC-5 was induced. Although NonO and PSF are nuclear paraspeckle components, these proteins formed cytoplasmic granules with HERMES in the neurites. G3BP1, a component of stress granules, was also colocalized to the granules, interacting with NonO and HERMES even in the absence of cellular stress. Consistent with a previous report that KIF5 interacts with neuronal granules, the localization of KIF5A overlapped with the cytoplasmic granules in differentiated RGC-5 cells. Thus, our study strongly suggests that the cytoplasmic granule containing HERMES, NonO, PSF, and G3BP1 is a neuronal RNA-protein granule that is transported in neurites during retinal differentiation. PMID:25651939

  20. Skipping the co-expression problem: the new 2A "CHYSEL" technology.

    PubMed

    de Felipe, Pablo

    2004-09-13

    The rapid progress in the field of genomics is increasing our knowledge of multi-gene diseases. However, any realistic hope of gene therapy treatment for those diseases needs first to address the problem of co-ordinately co-expressing several transgenes. Currently, the use of internal ribosomal entry sites (IRESs) is the strategy chosen by many researchers to ensure co-expression. The large sizes of the IRESs (~0.5 kb), and the difficulties of ensuring a well-balanced co-expression, have prompted several researchers to imitate a co-expression strategy used by many viruses: to express several proteins as a polyprotein. A small peptide of 18 amino acids (2A) from the foot-and-mouth disease virus (FMDV) is being used to avoid the need of proteinases to process the polyprotein. FMDV 2A is introduced as a linker between two proteins to allow autonomous intra-ribosomal self-processing of polyproteins. Recent reports have shown that this sequence is compatible with different sub-cellular targeting signals and can be used to co-express up to four proteins from a single retroviral vector. This short peptide provides a tool to allow the co-expression of multiple proteins from a single vector, a useful technology for those working with heteromultimeric proteins, biochemical pathways or combined/synergistic phenomena. PMID:15363111

  1. ImmuCo: a database of gene co-expression in immune cells.

    PubMed

    Wang, Pingzhang; Qi, Huiying; Song, Shibin; Li, Shuang; Huang, Ningyu; Han, Wenling; Ma, Dalong

    2015-01-01

    Current gene co-expression databases and correlation networks do not support cell-specific analysis. Gene co-expression and expression correlation are subtly different phenomena, although both are likely to be functionally significant. Here, we report a new database, ImmuCo (http://immuco.bjmu.edu.cn), which is a cell-specific database that contains information about gene co-expression in immune cells, identifying co-expression and correlation between any two genes. The strength of co-expression of queried genes is indicated by signal values and detection calls, whereas expression correlation and strength are reflected by Pearson correlation coefficients. A scatter plot of the signal values is provided to directly illustrate the extent of co-expression and correlation. In addition, the database allows the analysis of cell-specific gene expression profile across multiple experimental conditions and can generate a list of genes that are highly correlated with the queried genes. Currently, the database covers 18 human cell groups and 10 mouse cell groups, including 20,283 human genes and 20,963 mouse genes. More than 8.6 × 10(8) and 7.4 × 10(8) probe set combinations are provided for querying each human and mouse cell group, respectively. Sample applications support the distinctive advantages of the database. PMID:25326331

  2. [NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

    PubMed

    Posypanova, G A; Gaiduchenko, A I; Moskaleva, E Yu; Fedorov, G E

    2016-01-01

    The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons. PMID:27228654

  3. Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli.

    PubMed

    Liu, Xiang-Lei; Lin, Jun; Hu, Hai-Feng; Zhou, Bin; Zhu, Bao-Quan

    2016-04-01

    Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L(-1), which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3). PMID:27114316

  4. Co-Expression of Two Subtypes of Melatonin Receptor on Rat M1-Type Intrinsically Photosensitive Retinal Ganglion Cells

    PubMed Central

    Sheng, Wen-Long; Chen, Wei-Yi; Yang, Xiong-Li; Zhong, Yong-Mei; Weng, Shi-Jun

    2015-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions. PMID:25714375

  5. Elucidating gene function and function evolution through comparison of co-expression networks of plants

    PubMed Central

    Hansen, Bjoern O.; Vaid, Neha; Musialak-Lange, Magdalena; Janowski, Marcin; Mutwil, Marek

    2014-01-01

    The analysis of gene expression data has shown that transcriptionally coordinated (co-expressed) genes are often functionally related, enabling scientists to use expression data in gene function prediction. This Focused Review discusses our original paper (Large-scale co-expression approach to dissect secondary cell wall formation across plant species, Frontiers in Plant Science 2:23). In this paper we applied cross-species analysis to co-expression networks of genes involved in cellulose biosynthesis. We showed that the co-expression networks from different species are highly similar, indicating that whole biological pathways are conserved across species. This finding has two important implications. First, the analysis can transfer gene function annotation from well-studied plants, such as Arabidopsis, to other, uncharacterized plant species. As the analysis finds genes that have similar sequence and similar expression pattern across different organisms, functionally equivalent genes can be identified. Second, since co-expression analyses are often noisy, a comparative analysis should have higher performance, as parts of co-expression networks that are conserved are more likely to be functionally relevant. In this Focused Review, we outline the comparative analysis done in the original paper and comment on the recent advances and approaches that allow comparative analyses of co-function networks. We hypothesize that in comparison to simple co-expression analysis, comparative analysis would yield more accurate gene function predictions. Finally, by combining comparative analysis with genomic information of green plants, we propose a possible composition of cellulose biosynthesis machinery during earlier stages of plant evolution. PMID:25191328

  6. Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

    NASA Astrophysics Data System (ADS)

    Sanchez-Armass, Sergio; Sennoune, Souad; Martinez, Gloria M.; Ortega, Filiberta; Martinez-Zaguilan, Raul

    2002-06-01

    Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

  7. Analysis of bHLH coding genes using gene co-expression network approach.

    PubMed

    Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

    2016-07-01

    Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species. PMID:27178572

  8. Hi-C Chromatin Interaction Networks Predict Co-expression in the Mouse Cortex

    PubMed Central

    Hulsman, Marc; Lelieveldt, Boudewijn P. F.; de Ridder, Jeroen; Reinders, Marcel

    2015-01-01

    The three dimensional conformation of the genome in the cell nucleus influences important biological processes such as gene expression regulation. Recent studies have shown a strong correlation between chromatin interactions and gene co-expression. However, predicting gene co-expression from frequent long-range chromatin interactions remains challenging. We address this by characterizing the topology of the cortical chromatin interaction network using scale-aware topological measures. We demonstrate that based on these characterizations it is possible to accurately predict spatial co-expression between genes in the mouse cortex. Consistent with previous findings, we find that the chromatin interaction profile of a gene-pair is a good predictor of their spatial co-expression. However, the accuracy of the prediction can be substantially improved when chromatin interactions are described using scale-aware topological measures of the multi-resolution chromatin interaction network. We conclude that, for co-expression prediction, it is necessary to take into account different levels of chromatin interactions ranging from direct interaction between genes (i.e. small-scale) to chromatin compartment interactions (i.e. large-scale). PMID:25965262

  9. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  10. A novel method to identify pathways associated with renal cell carcinoma based on a gene co-expression network.

    PubMed

    Ruan, Xiyun; Li, Hongyun; Liu, Bo; Chen, Jie; Zhang, Shibao; Sun, Zeqiang; Liu, Shuangqing; Sun, Fahai; Liu, Qingyong

    2015-08-01

    The aim of the present study was to develop a novel method for identifying pathways associated with renal cell carcinoma (RCC) based on a gene co-expression network. A framework was established where a co-expression network was derived from the database as well as various co-expression approaches. First, the backbone of the network based on differentially expressed (DE) genes between RCC patients and normal controls was constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The differentially co-expressed links were detected by Pearson's correlation, the empirical Bayesian (EB) approach and Weighted Gene Co-expression Network Analysis (WGCNA). The co-expressed gene pairs were merged by a rank-based algorithm. We obtained 842; 371; 2,883 and 1,595 co-expressed gene pairs from the co-expression networks of the STRING database, Pearson's correlation EB method and WGCNA, respectively. Two hundred and eighty-one differentially co-expressed (DC) gene pairs were obtained from the merged network using this novel method. Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the network enrichment analysis (NEA) method were performed to verify feasibility of the merged method. Results of the KEGG and NEA pathway analyses showed that the network was associated with RCC. The suggested method was computationally efficient to identify pathways associated with RCC and has been identified as a useful complement to traditional co-expression analysis. PMID:26058425

  11. Intrinsic Cholinergic Neurons in the Hippocampus: Fact or Artifact?

    PubMed Central

    Blusztajn, Jan Krzysztof; Rinnofner, Jasmine

    2016-01-01

    It is generally agreed that hippocampal acetylcholine (ACh) is synthesized and released exclusively from the terminals of the long-axon afferents whose cell bodies reside in the medial septum and diagonal band. The search for intrinsic cholinergic neurons in the hippocampus has a long history; however evidence for the existence of these neurons has been inconsistent, with most investigators failing to detect them using in situ hybridization or immunohistochemical staining of the cholinergic markers, choline acetyltransferase (ChAT) or vesicular acetylcholine transporter (VAChT). Advances in the use of bacterial artificial chromosome (BAC) transgenic mice expressing a reporter protein under the control of the genomic elements of the Chat gene (Chat-BAC mice) have facilitated studies of cholinergic neurons. Such mice show robust and faithful expression of the reporter proteins in all known cholinergic cell populations. The availability of the Chat-BAC mice re-ignited interest in hippocampal cholinergic interneurons, because a small number of such reporter-expressing cells is frequently observed in the hippocampus of these mice. However, to date, attempts to confirm that these neurons co-express the endogenous cholinergic marker ChAT, or release ACh, have been unsuccessful. Without such confirmatory evidence it is best to conclude that there are no cholinergic neurons in the hippocampus. Similar considerations apply to other BAC transgenic lines, whose utility as a discovery tool for cell populations heretofore not known to express the genes of interest encoded by the BACs, must be validated by methods that detect expression of the endogenous genes. PMID:27014052

  12. Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury

    PubMed Central

    Eaton, Mary J.; Widerström-Noga, Eva; Wolfe, Stacey Quintero

    2011-01-01

    Transplant of cells which make biologic agents that can modulate the sensory and motor responses after spinal cord injury (SCI) would be useful to treat pain and paralysis. To address this need for clinically useful human cells, a unique neuronal cell line that synthesizes and secretes/releases the neurotransmitter serotonin (5HT) was isolated. Hind paw tactile allodynia and thermal hyperalgesia induced by severe contusive SCI were potently reversed after lumbar subarachnoid transplant of differentiated cells, but had no effect on open field motor scores, stride length, foot rotation, base of support, or gridwalk footfall errors associated with the SCI. The sensory effects appeared 1 week after transplant and did not diminish during the 8-week course of the experiment when grafts were placed 2 weeks after SCI. Many grafted cells were still present and synthesizing 5HT at the end of the study. These data suggest that the human neuronal serotonergic hNT2.19 cells can be used as a biologic minipump for receiving SCI-related neuropathic pain, but likely requires intraspinal grafts for motor recovery. PMID:21799949

  13. Human transcriptional interactome of chromatin contribute to gene co-expression

    PubMed Central

    2010-01-01

    Background Transcriptional interactome of chromatin is one of the important mechanisms in gene transcription regulation. By chromatin conformation capture and 3D FISH experiments, several chromatin interactions cases among sequence-distant genes or even inter-chromatin genes were reported. However, on genomics level, there is still little evidence to support these mechanisms. Recently based on Hi-C experiment, a genome-wide picture of chromatin interactions in human cells was presented. It provides a useful material for analysing whether the mechanism of transcriptional interactome is common. Results The main work here is to demonstrate whether the effects of transcriptional interactome on gene co-expression exist on genomic level. While controlling the effects of transcription factors control similarities (TCS), we tested the correlation between Hi-C interaction and the mutual ranks of gene co-expression rates (provided by COXPRESdb) of intra-chromatin gene pairs. We used 6,084 genes with both TF annotation and co-expression information, and matched them into 273,458 pairs with similar Hi-C interaction ranks in different cell types. The results illustrate that co-expression is strongly associated with chromatin interaction. Further analysis using GO annotation reveals potential correlation between gene function similarity, Hi-C interaction and their co-expression. Conclusions According to the results in this research, the intra-chromatin interactome may have relation to gene function and associate with co-expression. This study provides evidence for illustrating the effect of transcriptional interactome on transcription regulation. PMID:21156067

  14. Identification of hub genes and pathways associated with retinoblastoma based on co-expression network analysis.

    PubMed

    Wang, Q L; Chen, X; Zhang, M H; Shen, Q H; Qin, Z M

    2015-01-01

    The objective of this paper was to identify hub genes and pathways associated with retinoblastoma using centrality analysis of the co-expression network and pathway-enrichment analysis. The co-expression network of retinoblastoma was constructed by weighted gene co-expression network analysis (WGCNA) based on differentially expressed (DE) genes, and clusters were obtained through the molecular complex detection (MCODE) algorithm. Degree centrality analysis of the co-expression network was performed to explore hub genes present in retinoblastoma. Pathway-enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Validation of hub gene expression in retinoblastoma was performed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The co-expression network based on 221 DE genes between retinoblastoma and normal controls consisted of 210 nodes and 3965 edges, and 5 clusters of the network were evaluated. By assessing the centrality analysis of the co-expression network, 21 hub genes were identified, such as SNORD115-41, RASSF2, and SNORD115-44. According to RT-PCR analysis, 16 of the 21 hub genes were differently expressed, including RASSF2 and CDCA7, and 5 were not differently expressed in retinoblastoma compared to normal controls. Pathway analysis showed that genes in 2 clusters were enriched in 3 pathways: purine metabolism, p53 signaling pathway, and melanogenesis. In this study, we successfully identified 16 hub genes and 3 pathways associated with retinoblastoma, which may be potential biomarkers for early detection and therapy for retinoblastoma. PMID:26662407

  15. Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase Calpha and beta.

    PubMed

    Chen, Jun; Liu, Ying; Soh, Jae-Won; Aguilera, Greti

    2009-08-01

    Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway. PMID:19519660

  16. Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase C alpha and beta

    PubMed Central

    Chen, Jun; Liu, Ying; Soh, Jae-Won; Aguilera, Greti

    2009-01-01

    Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen activated protein (MAP) kinase-mediated Bad phosphorylation. Here we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP induced antiapoptosis in H32 cells. Serum deprivation increased PKCδ but not PKCα or PKCβ activity, while VP increased PKCα and PKCβ without affecting PKCδ activity. Inhibition of PKCδ prevented caspase-3 activation, indicating that PKCδ mediates the proapoptotic actions of serum deprivation. Simultaneous inhibition of PKC α&β and MAP kinase abolished VP-induced Bad phosphorylation, but it only partially prevented caspase-3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of PI3K/protein kinase B (Akt). The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCα&β together with ERK/MAP kinase activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway. PMID:19519660

  17. Single-Cell Co-expression Analysis Reveals Distinct Functional Modules, Co-regulation Mechanisms and Clinical Outcomes

    PubMed Central

    Wang, Jie; Xia, Shuli; Arand, Brian; Zhu, Heng; Machiraju, Raghu; Huang, Kun; Ji, Hongkai; Qian, Jiang

    2016-01-01

    Co-expression analysis has been employed to predict gene function, identify functional modules, and determine tumor subtypes. Previous co-expression analysis was mainly conducted at bulk tissue level. It is unclear whether co-expression analysis at the single-cell level will provide novel insights into transcriptional regulation. Here we developed a computational approach to compare glioblastoma expression profiles at the single-cell level with those obtained from bulk tumors. We found that the co-expressed genes observed in single cells and bulk tumors have little overlap and show distinct characteristics. The co-expressed genes identified in bulk tumors tend to have similar biological functions, and are enriched for intrachromosomal interactions with synchronized promoter activity. In contrast, single-cell co-expressed genes are enriched for known protein-protein interactions, and are regulated through interchromosomal interactions. Moreover, gene members of some protein complexes are co-expressed only at the bulk level, while those of other complexes are co-expressed at both single-cell and bulk levels. Finally, we identified a set of co-expressed genes that can predict the survival of glioblastoma patients. Our study highlights that comparative analyses of single-cell and bulk gene expression profiles enable us to identify functional modules that are regulated at different levels and hold great translational potential. PMID:27100869

  18. The Detection of Metabolite-Mediated Gene Module Co-Expression Using Multivariate Linear Models

    PubMed Central

    Padayachee, Trishanta; Khamiakova, Tatsiana; Shkedy, Ziv; Perola, Markus; Salo, Perttu; Burzykowski, Tomasz

    2016-01-01

    Investigating whether metabolites regulate the co-expression of a predefined gene module is one of the relevant questions posed in the integrative analysis of metabolomic and transcriptomic data. This article concerns the integrative analysis of the two high-dimensional datasets by means of multivariate models and statistical tests for the dependence between metabolites and the co-expression of a gene module. The general linear model (GLM) for correlated data that we propose models the dependence between adjusted gene expression values through a block-diagonal variance-covariance structure formed by metabolic-subset specific general variance-covariance blocks. Performance of statistical tests for the inference of conditional co-expression are evaluated through a simulation study. The proposed methodology is applied to the gene expression data of the previously characterized lipid-leukocyte module. Our results show that the GLM approach improves on a previous approach by being less prone to the detection of spurious conditional co-expression. PMID:26918614

  19. Improved expression of recombinant human factor IX by co-expression of GGCX, VKOR and furin.

    PubMed

    Liu, Jianming; Jonebring, Anna; Hagström, Jonas; Nyström, Ann-Christin; Lövgren, Ann

    2014-04-01

    Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94% yield. PMID:24567122

  20. Genome-Wide Tissue-Specific Gene Expression, Co-expression and Regulation of Co-expressed Genes in Adult Nematode Ascaris suum

    PubMed Central

    Rosa, Bruce A.; Jasmer, Douglas P.; Mitreva, Makedonka

    2014-01-01

    Background Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Methodology/Principal Findings Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. Conclusions/Significance The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a

  1. CoExpNetViz: Comparative Co-Expression Networks Construction and Visualization Tool

    PubMed Central

    Tzfadia, Oren; Diels, Tim; De Meyer, Sam; Vandepoele, Klaas; Aharoni, Asaph; Van de Peer, Yves

    2016-01-01

    Motivation: Comparative transcriptomics is a common approach in functional gene discovery efforts. It allows for finding conserved co-expression patterns between orthologous genes in closely related plant species, suggesting that these genes potentially share similar function and regulation. Several efficient co-expression-based tools have been commonly used in plant research but most of these pipelines are limited to data from model systems, which greatly limit their utility. Moreover, in addition, none of the existing pipelines allow plant researchers to make use of their own unpublished gene expression data for performing a comparative co-expression analysis and generate multi-species co-expression networks. Results: We introduce CoExpNetViz, a computational tool that uses a set of query or “bait” genes as an input (chosen by the user) and a minimum of one pre-processed gene expression dataset. The CoExpNetViz algorithm proceeds in three main steps; (i) for every bait gene submitted, co-expression values are calculated using mutual information and Pearson correlation coefficients, (ii) non-bait (or target) genes are grouped based on cross-species orthology, and (iii) output files are generated and results can be visualized as network graphs in Cytoscape. Availability: The CoExpNetViz tool is freely available both as a PHP web server (link: http://bioinformatics.psb.ugent.be/webtools/coexpr/) (implemented in C++) and as a Cytoscape plugin (implemented in Java). Both versions of the CoExpNetViz tool support LINUX and Windows platforms. PMID:26779228

  2. Differential co-expression analysis of venous thromboembolism based on gene expression profile data

    PubMed Central

    MING, ZHIBING; DING, WENBIN; YUAN, RUIFAN; JIN, JIE; LI, XIAOQIANG

    2016-01-01

    The aim of the present study was to screen differentially co-expressed genes and the involved transcription factors (TFs) and microRNAs (miRNAs) in venous thromboembolism (VTE). Microarray data of GSE19151 were downloaded from Gene Expression Omnibus, including 70 patients with VTE and 63 healthy controls. Principal component analysis (PCA) was performed using R software. Differential co-expression analysis was performed using R, followed by screening of modules using Cytoscape. Functional annotation was performed using Database for Annotation, Visualization, and Integrated Discovery. Moreover, Fisher test was used to screen key TFs and miRNAs for the modules. PCA revealed the disease and healthy samples could not be distinguished at the gene expression level. A total of 4,796 upregulated differentially co-expressed genes (e.g. zinc finger protein 264, electron-transfer-flavoprotein, beta polypeptide and Janus kinase 2) and 3,629 downregulated differentially co-expressed genes (e.g. adenylate cyclase 7 and single-stranded DNA binding protein 2) were identified, which were further mined to obtain 17 and eight modules separately. Functional annotation revealed that the largest upregulated module was primarily associated with acetylation and the largest downregulated module was mainly involved in mitochondrion. Moreover, 48 TFs and 62 miRNA families were screened for the 17 upregulated modules, such as E2F transcription factor 4, miR-30 and miR-135 regulating the largest module. Conversely, 35 TFs and 18 miRNA families were identified for the 8 downregulated modules, including mitochondrial ribosomal protein S12 and miR-23 regulating the largest module. Differentially co-expressed genes regulated by TFs and miRNAs may jointly contribute to the abnormal acetylation and mitochondrion presentation in the progression of VTE. PMID:27284300

  3. Comparison of co-expression measures: mutual information, correlation, and model based indices

    PubMed Central

    2012-01-01

    Background Co-expression measures are often used to define networks among genes. Mutual information (MI) is often used as a generalized correlation measure. It is not clear how much MI adds beyond standard (robust) correlation measures or regression model based association measures. Further, it is important to assess what transformations of these and other co-expression measures lead to biologically meaningful modules (clusters of genes). Results We provide a comprehensive comparison between mutual information and several correlation measures in 8 empirical data sets and in simulations. We also study different approaches for transforming an adjacency matrix, e.g. using the topological overlap measure. Overall, we confirm close relationships between MI and correlation in all data sets which reflects the fact that most gene pairs satisfy linear or monotonic relationships. We discuss rare situations when the two measures disagree. We also compare correlation and MI based approaches when it comes to defining co-expression network modules. We show that a robust measure of correlation (the biweight midcorrelation transformed via the topological overlap transformation) leads to modules that are superior to MI based modules and maximal information coefficient (MIC) based modules in terms of gene ontology enrichment. We present a function that relates correlation to mutual information which can be used to approximate the mutual information from the corresponding correlation coefficient. We propose the use of polynomial or spline regression models as an alternative to MI for capturing non-linear relationships between quantitative variables. Conclusion The biweight midcorrelation outperforms MI in terms of elucidating gene pairwise relationships. Coupled with the topological overlap matrix transformation, it often leads to more significantly enriched co-expression modules. Spline and polynomial networks form attractive alternatives to MI in case of non-linear relationships

  4. Brief Report: Isogenic Induced Pluripotent Stem Cell Lines From an Adult With Mosaic Down Syndrome Model Accelerated Neuronal Ageing and Neurodegeneration

    PubMed Central

    Murray, Aoife; Letourneau, Audrey; Canzonetta, Claudia; Stathaki, Elisavet; Gimelli, Stefania; Sloan‐Bena, Frederique; Abrehart, Robert; Goh, Pollyanna; Lim, Shuhui; Baldo, Chiara; Dagna‐Bricarelli, Franca; Hannan, Saad; Mortensen, Martin; Ballard, David; Syndercombe Court, Denise; Fusaki, Noemi; Hasegawa, Mamoru; Smart, Trevor G.; Bishop, Cleo; Antonarakis, Stylianos E.

    2015-01-01

    Abstract Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well‐controlled cell model systems. We have developed a first nonintegration‐reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high‐resolution whole genome CGH‐array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high‐content microscopic analysis. Early differentiation shows an imbalance of the lineage‐specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC‐derived neurons show increased production of amyloid peptide‐containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21‐derived neurons show significantly higher number of DNA double‐strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. Stem Cells 2015;33:2077–2084 PMID:25694335

  5. Xanthurenic Acid Binds to Neuronal G-Protein-Coupled Receptors That Secondarily Activate Cationic Channels in the Cell Line NCB-20

    PubMed Central

    Taleb, Omar; Maammar, Mohammed; Brumaru, Daniel; Bourguignon, Jean-Jacques; Schmitt, Martine; Klein, Christian; Kemmel, Véronique; Maitre, Michel; Mensah-Nyagan, Ayikoe Guy

    2012-01-01

    Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca2+ concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. PMID:23139790

  6. Xanthurenic acid binds to neuronal G-protein-coupled receptors that secondarily activate cationic channels in the cell line NCB-20.

    PubMed

    Taleb, Omar; Maammar, Mohammed; Brumaru, Daniel; Bourguignon, Jean-Jacques; Schmitt, Martine; Klein, Christian; Kemmel, Véronique; Maitre, Michel; Mensah-Nyagan, Ayikoe Guy

    2012-01-01

    Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+) concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. PMID:23139790

  7. Role of VEGF and VEGFR2 Receptor in Reversal of ALS-CSF Induced Degeneration of NSC-34 Motor Neuron Cell Line.

    PubMed

    Vijayalakshmi, K; Ostwal, Piyush; Sumitha, R; Shruthi, S; Varghese, Anu Mary; Mishra, Poojashree; Manohari, S Gowri; Sagar, B C; Sathyaprabha, T N; Nalini, A; Raju, T R; Alladi, Phalguni Anand

    2015-01-01

    Vascular endothelial growth factor (VEGF), the well-known angiogenic factor is both neurotrophic and neuroprotective. Altered VEGF signalling is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal degenerative disease of motor neurons. We have shown earlier that VEGF protects NSC-34 motor neuronal cell line, when exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF). Here, we have investigated the consequences of ALS-CSF and VEGF supplementation on the VEGFR2 receptor and endogenous VEGF expression. ALS-CSF caused significant down-regulation of VEGFR2 as well as the Calbindin-D28K levels, but not endogenous VEGF. Exogenous supplementation restored the depletion of VEGFR2 and Calbindin-D28K with a concomitant up-regulation of endogenous VEGF. The up-regulated caspase 3 in the ALS-CSF group was reinstated to basal levels along with a significant reduction in the number of TUNEL-positive cells. Electron photomicrographs of ALS-CSF-exposed cells divulged presence of cytoplasmic vacuoles alongside severe damage to organelles like mitochondria, endoplasmic reticulum, etc. Substantial recovery of most of the damaged organelles was noted in response to VEGF supplementation. While the enhancement in endogenous VEGF levels highlights the autocrine functions, the up-regulation of VEGFR2 receptor emphasizes the paracrine functions of VEGF in modulating its neuroprotective effect against ALS-CSF. The revival of cellular organellar structure, increased calbindin expression and enhanced survival in response to VEGF supplementation consolidates the opinion that VEGF indeed has a therapeutic potential in sporadic ALS. PMID:24880751

  8. Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

    PubMed Central

    Marinova, Zoya; Walitza, Susanne; Grünblatt, Edna

    2014-01-01

    Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential

  9. N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents.

    PubMed

    Mazzoccoli, Carmela; Ruggieri, Vitalba; Tataranni, Tiziana; Agriesti, Francesca; Laurenzana, Ilaria; Fratello, Angelo; Capitanio, Nazzareno; Piccoli, Claudia

    2016-05-01

    Neuroblastoma is the most commonly extra-cranial solid tumor of childhood frequently diagnosed. The nervous system-specific metabolite N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA in neurons, it is among the most abundant metabolites present in the central nervous system (CNS) and appears to be involved in many CNS disorders. The functional significance of the high NAA concentration in the brain remains uncertain, but it confers to NAA a unique clinical significance exploited in magnetic resonance spectroscopy. In the current study, we show that treatment of SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA inhibits cell growth. This effect is partly due to enhanced apoptosis, shown by decrease of the anti-apoptotic factors survivin and Bcl-xL, and partly to arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1 and p27Kip1. Moreover, NAA-treated SH-SY5Y cells exhibited morphological changes accompanied with increase of the neurogenic markers TH and MAP2 and down-regulation of the pluripotency markers OCT4 and CXCR4/CD184. Finally, NAA-pre-treated SH-SY5Y cells resulted more sensitive to the cytotoxic effect of the chemotherapeutic drugs Cisplatin and 5-fluorouracil.To our knowledge, this is the first study demonstrating the neuronal differentiating effects of NAA in neuroblastoma cells. NAA may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. PMID:27036033

  10. Apolipoprotein A-IV inhibits AgRP/NPY neurons and activates POMC neurons in the arcuate nucleus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However the mechanisms underlying its anorexigenic effects remain to be identified. We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and ne...

  11. Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

    PubMed Central

    Coppe, Alessandro; Ferrari, Francesco; Bisognin, Andrea; Danieli, Gian Antonio; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, Stefania

    2009-01-01

    Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation. PMID:19059999

  12. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7.

    PubMed

    Son, You Lee; Ubuka, Takayoshi; Soga, Tomoko; Yamamoto, Kazutoshi; Bentley, George E; Tsutsui, Kazuyoshi

    2016-06-01

    Gonadotropin-inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1-7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T-cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c-Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.-Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7. PMID:26929433

  13. VTCdb: a gene co-expression database for the crop species Vitis vinifera (grapevine)

    PubMed Central

    2013-01-01

    Background Gene expression datasets in model plants such as Arabidopsis have contributed to our understanding of gene function and how a single underlying biological process can be governed by a diverse network of genes. The accumulation of publicly available microarray data encompassing a wide range of biological and environmental conditions has enabled the development of additional capabilities including gene co-expression analysis (GCA). GCA is based on the understanding that genes encoding proteins involved in similar and/or related biological processes may exhibit comparable expression patterns over a range of experimental conditions, developmental stages and tissues. We present an open access database for the investigation of gene co-expression networks within the cultivated grapevine, Vitis vinifera. Description The new gene co-expression database, VTCdb (http://vtcdb.adelaide.edu.au/Home.aspx), offers an online platform for transcriptional regulatory inference in the cultivated grapevine. Using condition-independent and condition-dependent approaches, grapevine co-expression networks were constructed using the latest publicly available microarray datasets from diverse experimental series, utilising the Affymetrix Vitis vinifera GeneChip (16 K) and the NimbleGen Grape Whole-genome microarray chip (29 K), thus making it possible to profile approximately 29,000 genes (95% of the predicted grapevine transcriptome). Applications available with the online platform include the use of gene names, probesets, modules or biological processes to query the co-expression networks, with the option to choose between Affymetrix or Nimblegen datasets and between multiple co-expression measures. Alternatively, the user can browse existing network modules using interactive network visualisation and analysis via CytoscapeWeb. To demonstrate the utility of the database, we present examples from three fundamental biological processes (berry development, photosynthesis and

  14. Co-expression network-based analysis of hippocampal expression data associated with Alzheimer's disease using a novel algorithm

    PubMed Central

    YUE, HONG; YANG, BO; YANG, FANG; HU, XIAO-LI; KONG, FAN-BIN

    2016-01-01

    Recent progress in bioinformatics has facilitated the clarification of biological processes associated with complex diseases. Numerous methods of co-expression analysis have been proposed for use in the study of pairwise relationships among genes. In the present study, a combined network based on gene pairs was constructed following the conversion and combination of gene pair score values using a novel algorithm across multiple approaches. Three hippocampal expression profiles of patients with Alzheimer's disease (AD) and normal controls were extracted from the ArrayExpress database, and a total of 144 differentially expressed (DE) genes across multiple studies were identified by a rank product (RP) method. Five groups of co-expression gene pairs and five networks were identified and constructed using four existing methods [weighted gene co-expression network analysis (WGCNA), empirical Bayesian (EB), differentially co-expressed genes and links (DCGL), search tool for the retrieval of interacting genes/proteins database (STRING)] and a novel rank-based algorithm with combined score, respectively. Topological analysis indicated that the co-expression network constructed by the WGCNA method had the tendency to exhibit small-world characteristics, and the combined co-expression network was confirmed to be a scale-free network. Functional analysis of the co-expression gene pairs was conducted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The co-expression gene pairs were mostly enriched in five pathways, namely proteasome, oxidative phosphorylation, Parkinson's disease, Huntington's disease and AD. This study provides a new perspective to co-expression analysis. Since different methods of analysis often present varying abilities, the novel combination algorithm may provide a more credible and robust outcome, and could be used to complement to traditional co-expression analysis. PMID:27168792

  15. A novel method to identify pathways associated with renal cell carcinoma based on a gene co-expression network

    PubMed Central

    RUAN, XIYUN; LI, HONGYUN; LIU, BO; CHEN, JIE; ZHANG, SHIBAO; SUN, ZEQIANG; LIU, SHUANGQING; SUN, FAHAI; LIU, QINGYONG

    2015-01-01

    The aim of the present study was to develop a novel method for identifying pathways associated with renal cell carcinoma (RCC) based on a gene co-expression network. A framework was established where a co-expression network was derived from the database as well as various co-expression approaches. First, the backbone of the network based on differentially expressed (DE) genes between RCC patients and normal controls was constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The differentially co-expressed links were detected by Pearson’s correlation, the empirical Bayesian (EB) approach and Weighted Gene Co-expression Network Analysis (WGCNA). The co-expressed gene pairs were merged by a rank-based algorithm. We obtained 842; 371; 2,883 and 1,595 co-expressed gene pairs from the co-expression networks of the STRING database, Pearson’s correlation EB method and WGCNA, respectively. Two hundred and eighty-one differentially co-expressed (DC) gene pairs were obtained from the merged network using this novel method. Pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the network enrichment analysis (NEA) method were performed to verify feasibility of the merged method. Results of the KEGG and NEA pathway analyses showed that the network was associated with RCC. The suggested method was computationally efficient to identify pathways associated with RCC and has been identified as a useful complement to traditional co-expression analysis. PMID:26058425

  16. Vestibular Neuronitis

    MedlinePlus

    ... Prevent Painful Swimmer's Ear Additional Content Medical News Vestibular Neuronitis By Lawrence R. Lustig, MD NOTE: This ... Drugs Herpes Zoster Oticus Meniere Disease Purulent Labyrinthitis Vestibular Neuronitis Vestibular neuronitis is a disorder characterized by ...

  17. Dysregulation of system xc- expression induced by mutant huntingtin in a striatal neuronal cell line and in R6/2 mice

    PubMed Central

    Frederick, Natalie M.; Bertho, Julie; Patel, Kishan K.; Petr, Geraldine T.; Bakradze, Ekaterina; Smith, Sylvia B.; Rosenberg, Paul A.

    2014-01-01

    Oxidative stress has been implicated in the pathogenesis of Huntington's disease (HD), however, the origin of the oxidative stress is unknown. System xc- plays a role in the import of cystine to synthesize the antioxidant glutathione. We found in the STHdhQ7/Q7 and STHdhQ111/Q111 striatal cell lines, derived from neuronal precursor cells isolated from knock-in mice containing 7 or 111 CAG repeats in the huntingtin gene, that there is a decrease in system xc- function. System xc- is composed of two proteins, the substrate specific transporter, xCT, and an anchoring protein, CD98. The decrease in function in system xc- that we observed is caused by a decrease in xCT mRNA and protein expression in the STHdhQ111/Q111 cells. In addition we found a decrease in protein and mRNA expression in the transgenic R6/2 HD mouse model at 6 weeks of age. STHdhQ111/Q111 cells have lower basal levels of GSH and higher basal levels of ROS. Acute inhibition of system xc- causes greater increase in oxidative stress in the STHdhQ111/Q111 cells than in the STHdhQ7/Q7 cells. These results suggest that a defect in the regulation of xCT may be involved in the pathogenesis of HD by compromising xCT expression and increasing susceptibility to oxidative stress. PMID:25004085

  18. Differential effects of "Advanced glycation endproducts" and beta-amyloid peptide on glucose utilization and ATP levels in the neuronal cell line SH-SY5Y.

    PubMed

    Kuhla, B; Loske, C; Garcia De Arriba, S; Schinzel, R; Huber, J; Münch, G

    2004-03-01

    Beta-amyloid peptide (Abeta) and "Advanced glycation endproducts" (AGEs) are components of the senile plaques in Alzheimer's disease patients. It has been proposed that both AGEs and Abeta exert many of their effects, which include the upregulation of pro-inflammatory cytokines, through RAGE ("receptor for advanced glycation endproducts"). To investigate whether Abeta and AGEs cause similar or identical effects on cell survival and energy metabolism, we have compared the effects of a model-AGE and Abeta on cell viability, ATP level, glucose consumption and lactate production in the neuroblastoma cell line SH-SY5Y. The results show that AGEs and Abeta increase glucose consumption and decrease ATP levels in a dose dependent manner. Furthermore, both compounds decrease mitochondrial activity measured by the MTT assay. However, only AGEs decrease the number of cells and significantly increase lactate production. These data indicate that both AGEs and Abeta can cause differential disturbances in neuronal metabolism, which may contribute to the pathophysiological findings in Alzheimer's disease. However, their signalling pathways are apparently quite distinct, a fact which should stimulate a more detailed investigation in this field, e.g. for the purpose of a rational design of potential "neuroprotective" RAGE antagonists. PMID:14991463

  19. Strategies and Methodologies for the Co-expression of Multiple Proteins in Plants.

    PubMed

    Ferrer, Albert; Arró, Monserrat; Manzano, David; Altabella, Teresa

    2016-01-01

    The first transgenes were introduced in a plant genome more than 30 years ago. Since then, the capabilities of the plant scientific community to engineer the genome of plants have progressed at an unparalleled speed. Plant genetic engineering has become a central technology that has dramatically incremented our basic knowledge of plant biology and has enabled the translation of this knowledge into a number of increasingly complex and sophisticated biotechnological applications, which in most cases rely on the simultaneous co-expression of multiple recombinant proteins from different origins. To meet the new challenges of modern plant biotechnology, the plant scientific community has developed a vast arsenal of innovative molecular tools and genome engineering strategies. In this chapter we review a variety of tools, technologies, and strategies developed to transfer and simultaneously co-express multiple transgenes and proteins in a plant host. Their potential advantages, disadvantages, and future prospects are also discussed. PMID:27165331

  20. Characterization of Chemically Induced Liver Injuries Using Gene Co-Expression Modules

    PubMed Central

    Tawa, Gregory J.; AbdulHameed, Mohamed Diwan M.; Yu, Xueping; Kumar, Kamal; Ippolito, Danielle L.; Lewis, John A.; Stallings, Jonathan D.; Wallqvist, Anders

    2014-01-01

    Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules) specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1) known biochemical pathways associated with liver injuries and 2) clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20%) genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects. PMID:25226513

  1. An extensive (co-)expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

    PubMed Central

    Ehlting, Jürgen; Sauveplane, Vincent; Olry, Alexandre; Ginglinger, Jean-François; Provart, Nicholas J; Werck-Reichhart, Danièle

    2008-01-01

    Background Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s) is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. Results We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. Conclusion The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling. PMID:18433503

  2. Genome-wide patterns of promoter sharing and co-expression in bovine skeletal muscle

    PubMed Central

    2011-01-01

    Background Gene regulation by transcription factors (TF) is species, tissue and time specific. To better understand how the genetic code controls gene expression in bovine muscle we associated gene expression data from developing Longissimus thoracis et lumborum skeletal muscle with bovine promoter sequence information. Results We created a highly conserved genome-wide promoter landscape comprising 87,408 interactions relating 333 TFs with their 9,242 predicted target genes (TGs). We discovered that the complete set of predicted TGs share an average of 2.75 predicted TF binding sites (TFBSs) and that the average co-expression between a TF and its predicted TGs is higher than the average co-expression between the same TF and all genes. Conversely, pairs of TFs sharing predicted TGs showed a co-expression correlation higher that pairs of TFs not sharing TGs. Finally, we exploited the co-occurrence of predicted TFBS in the context of muscle-derived functionally-coherent modules including cell cycle, mitochondria, immune system, fat metabolism, muscle/glycolysis, and ribosome. Our findings enabled us to reverse engineer a regulatory network of core processes, and correctly identified the involvement of E2F1, GATA2 and NFKB1 in the regulation of cell cycle, fat, and muscle/glycolysis, respectively. Conclusion The pivotal implication of our research is two-fold: (1) there exists a robust genome-wide expression signal between TFs and their predicted TGs in cattle muscle consistent with the extent of promoter sharing; and (2) this signal can be exploited to recover the cellular mechanisms underpinning transcription regulation of muscle structure and development in bovine. Our study represents the first genome-wide report linking tissue specific co-expression to co-regulation in a non-model vertebrate. PMID:21226902

  3. Massive-Scale Gene Co-Expression Network Construction and Robustness Testing Using Random Matrix Theory

    PubMed Central

    Isaacson, Sven; Luo, Feng; Feltus, Frank A.; Smith, Melissa C.

    2013-01-01

    The study of gene relationships and their effect on biological function and phenotype is a focal point in systems biology. Gene co-expression networks built using microarray expression profiles are one technique for discovering and interpreting gene relationships. A knowledge-independent thresholding technique, such as Random Matrix Theory (RMT), is useful for identifying meaningful relationships. Highly connected genes in the thresholded network are then grouped into modules that provide insight into their collective functionality. While it has been shown that co-expression networks are biologically relevant, it has not been determined to what extent any given network is functionally robust given perturbations in the input sample set. For such a test, hundreds of networks are needed and hence a tool to rapidly construct these networks. To examine functional robustness of networks with varying input, we enhanced an existing RMT implementation for improved scalability and tested functional robustness of human (Homo sapiens), rice (Oryza sativa) and budding yeast (Saccharomyces cerevisiae). We demonstrate dramatic decrease in network construction time and computational requirements and show that despite some variation in global properties between networks, functional similarity remains high. Moreover, the biological function captured by co-expression networks thresholded by RMT is highly robust. PMID:23409071

  4. Co-expression network analysis identifies transcriptional modules in the mouse liver.

    PubMed

    Liu, Wei; Ye, Hua

    2014-10-01

    The mouse liver transcriptome has been extensively studied but little is known about the global hepatic gene network of the mouse under normal physiological conditions. Understanding this will help reveal the transcriptional organization of the liver and elucidate its functional complexity. Here, weighted gene co-expression network analysis (WGCNA) was carried out to explore gene co-expression networks using large-scale microarray data from normal mouse livers. A total of 7,203 genes were parsed into 16 gene modules associated with protein catabolism, RNA processing, muscle contraction, transcriptional regulation, oxidation reduction, sterol biosynthesis, translation, fatty acid metabolism, immune response and others. The modules were organized into higher order co-expression groups. Hub genes in each module were found to be critical for module function. In sum, the analyses revealed the gene modular map of the mouse liver under normal physiological condition. These results provide a systems-level framework to help understand the complexity of the mouse liver at the molecular level, and should be beneficial in annotating uncharacterized genes. PMID:24816893

  5. WeGET: predicting new genes for molecular systems by weighted co-expression.

    PubMed

    Szklarczyk, Radek; Megchelenbrink, Wout; Cizek, Pavel; Ledent, Marie; Velemans, Gonny; Szklarczyk, Damian; Huynen, Martijn A

    2016-01-01

    We have developed the Weighted Gene Expression Tool and database (WeGET, http://weget.cmbi.umcn.nl) for the prediction of new genes of a molecular system by correlated gene expression. WeGET utilizes a compendium of 465 human and 560 murine gene expression datasets that have been collected from multiple tissues under a wide range of experimental conditions. It exploits this abundance of expression data by assigning a high weight to datasets in which the known genes of a molecular system are harmoniously up- and down-regulated. WeGET ranks new candidate genes by calculating their weighted co-expression with that system. A weighted rank is calculated for human genes and their mouse orthologs. Then, an integrated gene rank and p-value is computed using a rank-order statistic. We applied our method to predict novel genes that have a high degree of co-expression with Gene Ontology terms and pathways from KEGG and Reactome. For each query set we provide a list of predicted novel genes, computed weights for transcription datasets used and cell and tissue types that contributed to the final predictions. The performance for each query set is assessed by 10-fold cross-validation. Finally, users can use the WeGET to predict novel genes that co-express with a custom query set. PMID:26582928

  6. Context Specific and Differential Gene Co-expression Networks via Bayesian Biclustering.

    PubMed

    Gao, Chuan; McDowell, Ian C; Zhao, Shiwen; Brown, Christopher D; Engelhardt, Barbara E

    2016-07-01

    Identifying latent structure in high-dimensional genomic data is essential for exploring biological processes. Here, we consider recovering gene co-expression networks from gene expression data, where each network encodes relationships between genes that are co-regulated by shared biological mechanisms. To do this, we develop a Bayesian statistical model for biclustering to infer subsets of co-regulated genes that covary in all of the samples or in only a subset of the samples. Our biclustering method, BicMix, allows overcomplete representations of the data, computational tractability, and joint modeling of unknown confounders and biological signals. Compared with related biclustering methods, BicMix recovers latent structure with higher precision across diverse simulation scenarios as compared to state-of-the-art biclustering methods. Further, we develop a principled method to recover context specific gene co-expression networks from the estimated sparse biclustering matrices. We apply BicMix to breast cancer gene expression data and to gene expression data from a cardiovascular study cohort, and we recover gene co-expression networks that are differential across ER+ and ER- samples and across male and female samples. We apply BicMix to the Genotype-Tissue Expression (GTEx) pilot data, and we find tissue specific gene networks. We validate these findings by using our tissue specific networks to identify trans-eQTLs specific to one of four primary tissues. PMID:27467526

  7. WeGET: predicting new genes for molecular systems by weighted co-expression

    PubMed Central

    Szklarczyk, Radek; Megchelenbrink, Wout; Cizek, Pavel; Ledent, Marie; Velemans, Gonny; Szklarczyk, Damian; Huynen, Martijn A.

    2016-01-01

    We have developed the Weighted Gene Expression Tool and database (WeGET, http://weget.cmbi.umcn.nl) for the prediction of new genes of a molecular system by correlated gene expression. WeGET utilizes a compendium of 465 human and 560 murine gene expression datasets that have been collected from multiple tissues under a wide range of experimental conditions. It exploits this abundance of expression data by assigning a high weight to datasets in which the known genes of a molecular system are harmoniously up- and down-regulated. WeGET ranks new candidate genes by calculating their weighted co-expression with that system. A weighted rank is calculated for human genes and their mouse orthologs. Then, an integrated gene rank and p-value is computed using a rank-order statistic. We applied our method to predict novel genes that have a high degree of co-expression with Gene Ontology terms and pathways from KEGG and Reactome. For each query set we provide a list of predicted novel genes, computed weights for transcription datasets used and cell and tissue types that contributed to the final predictions. The performance for each query set is assessed by 10-fold cross-validation. Finally, users can use the WeGET to predict novel genes that co-express with a custom query set. PMID:26582928

  8. Context Specific and Differential Gene Co-expression Networks via Bayesian Biclustering

    PubMed Central

    McDowell, Ian C.; Zhao, Shiwen; Brown, Christopher D.; Engelhardt, Barbara E.

    2016-01-01

    Identifying latent structure in high-dimensional genomic data is essential for exploring biological processes. Here, we consider recovering gene co-expression networks from gene expression data, where each network encodes relationships between genes that are co-regulated by shared biological mechanisms. To do this, we develop a Bayesian statistical model for biclustering to infer subsets of co-regulated genes that covary in all of the samples or in only a subset of the samples. Our biclustering method, BicMix, allows overcomplete representations of the data, computational tractability, and joint modeling of unknown confounders and biological signals. Compared with related biclustering methods, BicMix recovers latent structure with higher precision across diverse simulation scenarios as compared to state-of-the-art biclustering methods. Further, we develop a principled method to recover context specific gene co-expression networks from the estimated sparse biclustering matrices. We apply BicMix to breast cancer gene expression data and to gene expression data from a cardiovascular study cohort, and we recover gene co-expression networks that are differential across ER+ and ER- samples and across male and female samples. We apply BicMix to the Genotype-Tissue Expression (GTEx) pilot data, and we find tissue specific gene networks. We validate these findings by using our tissue specific networks to identify trans-eQTLs specific to one of four primary tissues. PMID:27467526

  9. Co-expression of soybean Dicer-like genes in response to stress and development.

    PubMed

    Curtin, Shaun J; Kantar, Michael B; Yoon, Han W; Whaley, Adam M; Schlueter, Jessica A; Stupar, Robert M

    2012-11-01

    Regulation of gene transcription and post-transcriptional processes is critical for proper development, genome integrity, and stress responses in plants. Many genes involved in the key processes of transcriptional and post-transcriptional regulation have been well studied in model diploid organisms. However, gene and genome duplication may alter the function of the genes involved in these processes. To address this question, we assayed the stress-induced transcription patterns of duplicated gene pairs involved in RNAi and DNA methylation processes in the paleopolyploid soybean. Real-time quantitative PCR and Sequenom MassARRAY expression assays were used to profile the relative expression ratios of eight gene pairs across eight different biotic and abiotic stress conditions. The transcriptional responses to stress for genes involved in DNA methylation, RNAi processing, and miRNA processing were compared. The strongest evidence for pairwise co-expression in response to stresses was exhibited by non-paralogous Dicer-like (DCL) genes GmDCL2a-GmDCL3a and GmDCL1b-GmDCL2b, most profoundly in root tissues. Among homoeologous or paralogous DCL genes, the Dicer-like 2 (DCL2) gene pair exhibited the strongest response to stress and most conserved co-expression pattern. This was surprising because the DCL2 duplication event is more ancient than the other DCL duplications. Possible mechanisms that may be driving the DCL2 co-expression are discussed. PMID:22527487

  10. Reconstruction of gene co-expression network from microarray data using local expression patterns

    PubMed Central

    2014-01-01

    Background Biological networks connect genes, gene products to one another. A network of co-regulated genes may form gene clusters that can encode proteins and take part in common biological processes. A gene co-expression network describes inter-relationships among genes. Existing techniques generally depend on proximity measures based on global similarity to draw the relationship between genes. It has been observed that expression profiles are sharing local similarity rather than global similarity. We propose an expression pattern based method called GeCON to extract Gene CO-expression Network from microarray data. Pair-wise supports are computed for each pair of genes based on changing tendencies and regulation patterns of the gene expression. Gene pairs showing negative or positive co-regulation under a given number of conditions are used to construct such gene co-expression network. We construct co-expression network with signed edges to reflect up- and down-regulation between pairs of genes. Most existing techniques do not emphasize computational efficiency. We exploit a fast correlogram matrix based technique for capturing the support of each gene pair to construct the network. Results We apply GeCON to both real and synthetic gene expression data. We compare our results using the DREAM (Dialogue for Reverse Engineering Assessments and Methods) Challenge data with three well known algorithms, viz., ARACNE, CLR and MRNET. Our method outperforms other algorithms based on in silico regulatory network reconstruction. Experimental results show that GeCON can extract functionally enriched network modules from real expression data. Conclusions In view of the results over several in-silico and real expression datasets, the proposed GeCON shows satisfactory performance in predicting co-expression network in a computationally inexpensive way. We further establish that a simple expression pattern matching is helpful in finding biologically relevant gene network. In

  11. Annotation of gene function in citrus using gene expression information and co-expression networks

    PubMed Central

    2014-01-01

    Background The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. Results We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Conclusions Integration of citrus gene co-expression networks

  12. Long-term survival and differentiation of retinal neurons derived from human embryonic stem cell lines in un-immunosuppressed mouse retina

    PubMed Central

    Hambright, Dustin; Park, Kye-Yoon; Brooks, Matthew; McKay, Ron; Swaroop, Anand

    2012-01-01

    Purpose To examine the potential of NIH-maintained human embryonic stem cell (hESC) lines TE03 and UC06 to differentiate into retinal progenitor cells (hESC-RPCs) using the noggin/Dkk-1/IGF-1/FGF9 protocol. An additional goal is to examine the in vivo dynamics of maturation and retinal integration of subretinal and epiretinal (vitreous space) hESC-RPC grafts without immunosuppression. Methods hESCs were neuralized in vitro with noggin for 2 weeks and expanded to derive neuroepithelial cells (hESC-neural precursors, NPs). Wnt (Integration 1 and wingless) blocking morphogens Dickkopf-1 (Dkk-1) and Insulin-like growth factor 1 (IGF-1) were used to direct NPs to a rostral neural fate, and fibroblast growth factor 9 (FGF9)/fibroblast growth factor-basic (bFGF) were added to bias the differentiation of developing anterior neuroectoderm cells to neural retina (NR) rather than retinal pigment epithelium (RPE). Cells were dissociated and grafted into the subretinal and epiretinal space of young adult (4–6-week-old) mice (C57BL/6J x129/Sv mixed background). Remaining cells were replated for (i) immunocytochemical analysis and (ii) used for quantitative reverse transcription polymerase chain reaction (qRT–PCR) analysis. Mice were sacrificed 3 weeks or 3 months after grafting, and the grafts were examined by histology and immunohistochemistry for survival of hESC-RPCs, presence of mature neuronal and retinal markers, and the dynamics of in vivo maturation and integration into the host retina. Results At the time of grafting, hESC-RPCs exhibited immature neural/neuronal immunophenotypes represented by nestin and neuronal class III β-tubulin, with about half of the cells positive for cell proliferation marker Kiel University -raised antibody number 67 (Ki67), and no recoverin-positive (recoverin [+]) cells. The grafted cells expressed eye field markers paired box 6 (PAX6), retina and anterior neural fold homeobox (RAX), sine oculis homeobox homolog 6 (SIX6), LIM homeobox 2

  13. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    PubMed Central

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops. PMID:26528311

  14. The protective effect of astrocyte-derived 14,15-EET on H2O2-induced cell injury in Astrocyte-dopaminergic neuronal cell line co-culture

    PubMed Central

    Terashvili, Maia; Sarkar, Pallabi; Van Nostrand, Meg; Falck, John R.; Harder, David R.

    2014-01-01

    Astrocytes perform several functions that are essential for normal neuronal activity. They play a critical role in neuronal survival during ischemia and other degenerative injuries and also modulate neuronal recovery by influencing neurite outgrowth. In this study, we investigated the neuroprotective effects of astrocyte-derived 14,15-epoxyeicosatrienoic acid (14,15-EET), metabolite of arachidonic acid by Cytochrome P450 epoxygenases (CYP), against oxidative stress induced by hydrogen peroxide (H2O2). We found that dopaminergic neuronal cells (N27 cell line) stimulated with two different doses of H2O2 (0.1 and 1 mM) for 1h showed decreased cell viability compared to the control group, while astrocytes co-cultured with dopaminergic neuronal cell lines prevented cell during after stimulation with the same doses of H2O2 for 1h. Dopaminergic neuronal cells (N27 cell line) pretreated with different doses of 14, 15-EET (0.1–30 μM, 30 min) before H2O2 stimulation also showed increased cell viability. Furthermore, pre-treatment of the co-cultured cells with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of the EET metabolizing enzyme, soluble epoxide hydrolase (sEH), before H2O2 stimulation (1 mM, for 1h) increased cell viability. It also increased the endogenous level of 14,15-EET in the media compared to control group. However, pretreatment with the CYP epoxygenase inhibitor miconazole (1–20 μM, 1h) before H2O2 (1 mM, 1h) stimulation showed decreased cell viability. Our data suggest that 14,15-EET which is released from astrocytes, enhances cell viability against oxidant induced injury. Further understanding of the mechanism of 14,15-EET-mediated protection in dopaminergic neurons is imperative, as it could lead to novel therapeutic approaches for treating CNS neuropathologies, such as Parkinson’s disease. PMID:22863680

  15. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

    PubMed Central

    Eaton, Mary J.; Berrocal, Yerko; Wolfe, Stacey Q.

    2012-01-01

    Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain. PMID:22619713

  16. Co-expression analysis reveals a group of genes potentially involved in regulation of plant response to iron-deficiency.

    PubMed

    Li, Hua; Wang, Lei; Yang, Zhi Min

    2015-01-01

    Iron (Fe) is an essential element for plant growth and development. Iron deficiency results in abnormal metabolisms from respiration to photosynthesis. Exploration of Fe-deficient responsive genes and their networks is critically important to understand molecular mechanisms leading to the plant adaptation to soil Fe-limitation. Co-expression genes are a cluster of genes that have a similar expression pattern to execute relatively biological functions at a stage of development or under a certain environmental condition. They may share a common regulatory mechanism. In this study, we investigated Fe-starved-related co-expression genes from Arabidopsis. From the biological process GO annotation of TAIR (The Arabidopsis Information Resource), 180 iron-deficient responsive genes were detected. Using ATTED-II database, we generated six gene co-expression networks. Among these, two modules of PYE and IRT1 were successfully constructed. There are 30 co-expression genes that are incorporated in the two modules (12 in PYE-module and 18 in IRT1-module). Sixteen of the co-expression genes were well characterized. The remaining genes (14) are poorly or not functionally identified with iron stress. Validation of the 14 genes using real-time PCR showed differential expression under iron-deficiency. Most of the co-expression genes (23/30) could be validated in pye and fit mutant plants with iron-deficiency. We further identified iron-responsive cis-elements upstream of the co-expression genes and found that 22 out of 30 genes contain the iron-responsive motif IDE1. Furthermore, some auxin and ethylene-responsive elements were detected in the promoters of the co-expression genes. These results suggest that some of the genes can be also involved in iron stress response through the phytohormone-responsive pathways. PMID:25300251

  17. Conserved Units of Co-Expression in Bacterial Genomes: An Evolutionary Insight into Transcriptional Regulation

    PubMed Central

    Junier, Ivan; Rivoire, Olivier

    2016-01-01

    Genome-wide measurements of transcriptional activity in bacteria indicate that the transcription of successive genes is strongly correlated beyond the scale of operons. Here, we analyze hundreds of bacterial genomes to identify supra-operonic segments of genes that are proximal in a large number of genomes. We show that these synteny segments correspond to genomic units of strong transcriptional co-expression. Structurally, the segments contain operons with specific relative orientations (co-directional or divergent) and nucleoid-associated proteins are found to bind at their boundaries. Functionally, operons inside a same segment are highly co-expressed even in the apparent absence of regulatory factors at their promoter regions. Remote operons along DNA can also be co-expressed if their corresponding segments share a transcriptional or sigma factor, without requiring these factors to bind directly to the promoters of the operons. As evidence that these results apply across the bacterial kingdom, we demonstrate them both in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus subtilis. The underlying process that we propose involves only RNA-polymerases and DNA: it implies that the transcription of an operon mechanically enhances the transcription of adjacent operons. In support of a primary role of this regulation by facilitated co-transcription, we show that the transcription en bloc of successive operons as a result of transcriptional read-through is strongly and specifically enhanced in synteny segments. Finally, our analysis indicates that facilitated co-transcription may be evolutionary primitive and may apply beyond bacteria. PMID:27195891

  18. Co-expression of mitosis-regulating genes contributes to malignant progression and prognosis in oligodendrogliomas.

    PubMed

    Liu, Yanwei; Hu, Huimin; Zhang, Chuanbao; Wang, Haoyuan; Zhang, Wenlong; Wang, Zheng; Li, Mingyang; Zhang, Wei; Zhou, Dabiao; Jiang, Tao

    2015-11-10

    The clinical prognosis of patients with glioma is determined by tumor grades, but tumors of different subtypes with equal malignancy grade usually have different prognosis that is largely determined by genetic abnormalities. Oligodendrogliomas (ODs) are the second most common type of gliomas. In this study, integrative analyses found that distribution of TCGA transcriptomic subtypes was associated with grade progression in ODs. To identify critical gene(s) associated with tumor grades and TCGA subtypes, we analyzed 34 normal brain tissue (NBT), 146 WHO grade II and 130 grade III ODs by microarray and RNA sequencing, and identified a co-expression network of six genes (AURKA, NDC80, CENPK, KIAA0101, TIMELESS and MELK) that was associated with tumor grades and TCGA subtypes as well as Ki-67 expression. Validation of the six genes was performed by qPCR in additional 28 ODs. Importantly, these genes also were validated in four high-grade recurrent gliomas and the initial lower-grade gliomas resected from the same patients. Finally, the RNA data on two genes with the highest discrimination potential (AURKA and NDC80) and Ki-67 were validated on an independent cohort (5 NBTs and 86 ODs) by immunohistochemistry. Knockdown of AURKA and NDC80 by siRNAs suppressed Ki-67 expression and proliferation of gliomas cells. Survival analysis showed that high expression of the six genes corporately indicated a poor survival outcome. Correlation and protein interaction analysis provided further evidence for this co-expression network. These data suggest that the co-expression of the six mitosis-regulating genes was associated with malignant progression and prognosis in ODs. PMID:26468983

  19. Co-expression of mitosis-regulating genes contributes to malignant progression and prognosis in oligodendrogliomas

    PubMed Central

    Liu, Yanwei; Hu, Huimin; Zhang, Chuanbao; Wang, Haoyuan; Zhang, Wenlong; Wang, Zheng; Li, Mingyang; Zhang, Wei; Zhou, Dabiao; Jiang, Tao

    2015-01-01

    The clinical prognosis of patients with glioma is determined by tumor grades, but tumors of different subtypes with equal malignancy grade usually have different prognosis that is largely determined by genetic abnormalities. Oligodendrogliomas (ODs) are the second most common type of gliomas. In this study, integrative analyses found that distribution of TCGA transcriptomic subtypes was associated with grade progression in ODs. To identify critical gene(s) associated with tumor grades and TCGA subtypes, we analyzed 34 normal brain tissue (NBT), 146 WHO grade II and 130 grade III ODs by microarray and RNA sequencing, and identified a co-expression network of six genes (AURKA, NDC80,CENPK, KIAA0101, TIMELESS and MELK) that was associated with tumor grades and TCGA subtypes as well as Ki-67 expression. Validation of the six genes was performed by qPCR in additional 28 ODs. Importantly, these genes also were validated in four high-grade recurrent gliomas and the initial lower-grade gliomas resected from the same patients. Finally, the RNA data on two genes with the highest discrimination potential (AURKA and NDC80) and Ki-67 were validated on an independent cohort (5 NBTs and 86 ODs) by immunohistochemistry. Knockdown of AURKA and NDC80 by siRNAs suppressed Ki-67 expression and proliferation of gliomas cells. Survival analysis showed that high expression of the six genes corporately indicated a poor survival outcome. Correlation and protein interaction analysis provided further evidence for this co-expression network. These data suggest that the co-expression of the six mitosis-regulating genes was associated with malignant progression and prognosis in ODs. PMID:26468983

  20. Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons.

    PubMed

    Jensen, P; Ducray, A D; Widmer, H R; Meyer, M

    2015-12-01

    Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be

  1. Novel structural co-expression analysis linking the NPM1-associated ribosomal biogenesis network to chronic myelogenous leukemia

    PubMed Central

    Chan, Lawrence WC; Lin, Xihong; Yung, Godwin; Lui, Thomas; Chiu, Ya Ming; Wang, Fengfeng; Tsui, Nancy BY; Cho, William CS; Yip, SP; Siu, Parco M.; Wong, SC Cesar; Yung, Benjamin YM

    2015-01-01

    Co-expression analysis reveals useful dysregulation patterns of gene cooperativeness for understanding cancer biology and identifying new targets for treatment. We developed a structural strategy to identify co-expressed gene networks that are important for chronic myelogenous leukemia (CML). This strategy compared the distributions of expressional correlations between CML and normal states, and it identified a data-driven threshold to classify strongly co-expressed networks that had the best coherence with CML. Using this strategy, we found a transcriptome-wide reduction of co-expression connectivity in CML, reflecting potentially loosened molecular regulation. Conversely, when we focused on nucleophosmin 1 (NPM1) associated networks, NPM1 established more co-expression linkages with BCR-ABL pathways and ribosomal protein networks in CML than normal. This finding implicates a new role of NPM1 in conveying tumorigenic signals from the BCR-ABL oncoprotein to ribosome biogenesis, affecting cellular growth. Transcription factors may be regulators of the differential co-expression patterns between CML and normal. PMID:26205693

  2. Tissue-specific Co-expression of Long Non-coding and Coding RNAs Associated with Breast Cancer

    PubMed Central

    Wu, Wenting; Wagner, Erin K.; Hao, Yangyang; Rao, Xi; Dai, Hongji; Han, Jiali; Chen, Jinhui; Storniolo, Anna Maria V.; Liu, Yunlong; He, Chunyan

    2016-01-01

    Inference of the biological roles of lncRNAs in breast cancer development remains a challenge. Here, we analyzed RNA-seq data in tumor and normal breast tissue samples from 18 breast cancer patients and 18 healthy controls and constructed a functional lncRNA-mRNA co-expression network. We revealed two distinctive co-expression patterns associated with breast cancer, reflecting different underlying regulatory mechanisms: (1) 516 pairs of lncRNA-mRNAs have differential co-expression pattern, in which the correlation between lncRNA and mRNA expression differs in tumor and normal breast tissue; (2) 291 pairs have dose-response co-expression pattern, in which the correlation is similar, but the expression level of lncRNA or mRNA differs in the two tissue types. We further validated our findings in TCGA dataset and annotated lncRNAs using TANRIC. One novel lncRNA, AC145110.1 on 8p12, was found differentially co-expressed with 127 mRNAs (including TOX4 and MAEL) in tumor and normal breast tissue and also highly correlated with breast cancer clinical outcomes. Functional enrichment and pathway analyses identified distinct biological functions for different patterns of co-expression regulations. Our data suggested that lncRNAs might be involved in breast tumorigenesis through the modulation of gene expression in multiple pathologic pathways. PMID:27597120

  3. Tissue-specific Co-expression of Long Non-coding and Coding RNAs Associated with Breast Cancer.

    PubMed

    Wu, Wenting; Wagner, Erin K; Hao, Yangyang; Rao, Xi; Dai, Hongji; Han, Jiali; Chen, Jinhui; Storniolo, Anna Maria V; Liu, Yunlong; He, Chunyan

    2016-01-01

    Inference of the biological roles of lncRNAs in breast cancer development remains a challenge. Here, we analyzed RNA-seq data in tumor and normal breast tissue samples from 18 breast cancer patients and 18 healthy controls and constructed a functional lncRNA-mRNA co-expression network. We revealed two distinctive co-expression patterns associated with breast cancer, reflecting different underlying regulatory mechanisms: (1) 516 pairs of lncRNA-mRNAs have differential co-expression pattern, in which the correlation between lncRNA and mRNA expression differs in tumor and normal breast tissue; (2) 291 pairs have dose-response co-expression pattern, in which the correlation is similar, but the expression level of lncRNA or mRNA differs in the two tissue types. We further validated our findings in TCGA dataset and annotated lncRNAs using TANRIC. One novel lncRNA, AC145110.1 on 8p12, was found differentially co-expressed with 127 mRNAs (including TOX4 and MAEL) in tumor and normal breast tissue and also highly correlated with breast cancer clinical outcomes. Functional enrichment and pathway analyses identified distinct biological functions for different patterns of co-expression regulations. Our data suggested that lncRNAs might be involved in breast tumorigenesis through the modulation of gene expression in multiple pathologic pathways. PMID:27597120

  4. Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model.

    PubMed

    Hunter, Morag Rose; Grimsey, Natasha Lillia; Glass, Michelle

    2016-01-01

    G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags. PMID:27273047

  5. Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

    PubMed Central

    Hunter, Morag Rose; Grimsey, Natasha Lillia; Glass, Michelle

    2016-01-01

    G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags. PMID:27273047

  6. Opsin co-expression in Limulus photoreceptors: differential regulation by light and a circadian clock.

    PubMed

    Katti, C; Kempler, K; Porter, M L; Legg, A; Gonzalez, R; Garcia-Rivera, E; Dugger, D; Battelle, B-A

    2010-08-01

    A long-standing concept in vision science has held that a single photoreceptor expresses a single type of opsin, the protein component of visual pigment. However, the number of examples in the literature of photoreceptors from vertebrates and invertebrates that break this rule is increasing. Here, we describe a newly discovered Limulus opsin, Limulus opsin5, which is significantly different from previously characterized Limulus opsins, opsins1 and 2. We show that opsin5 is co-expressed with opsins1 and 2 in Limulus lateral and ventral eye photoreceptors and provide the first evidence that the expression of co-expressed opsins can be differentially regulated. We show that the relative levels of opsin5 and opsin1 and 2 in the rhabdom change with a diurnal rhythm and that their relative levels are also influenced by the animal's central circadian clock. An analysis of the sequence of opsin5 suggests it is sensitive to visible light (400-700 nm) but that its spectral properties may be different from that of opsins1 and 2. Changes in the relative levels of these opsins may underlie some of the dramatic day-night changes in Limulus photoreceptor function and may produce a diurnal change in their spectral sensitivity. PMID:20639420

  7. AuPairWise: A Method to Estimate RNA-Seq Replicability through Co-expression.

    PubMed

    Ballouz, Sara; Gillis, Jesse

    2016-04-01

    In addition to detecting novel transcripts and higher dynamic range, a principal claim for RNA-sequencing has been greater replicability, typically measured in sample-sample correlations of gene expression levels. Through a re-analysis of ENCODE data, we show that replicability of transcript abundances will provide misleading estimates of the replicability of conditional variation in transcript abundances (i.e., most expression experiments). Heuristics which implicitly address this problem have emerged in quality control measures to obtain 'good' differential expression results. However, these methods involve strict filters such as discarding low expressing genes or using technical replicates to remove discordant transcripts, and are costly or simply ad hoc. As an alternative, we model gene-level replicability of differential activity using co-expressing genes. We find that sets of housekeeping interactions provide a sensitive means of estimating the replicability of expression changes, where the co-expressing pair can be regarded as pseudo-replicates of one another. We model the effects of noise that perturbs a gene's expression within its usual distribution of values and show that perturbing expression by only 5% within that range is readily detectable (AUROC~0.73). We have made our method available as a set of easily implemented R scripts. PMID:27082953

  8. A co-expression modules based gene selection for cancer recognition.

    PubMed

    Lu, Xinguo; Deng, Yong; Huang, Lei; Feng, Bingtao; Liao, Bo

    2014-12-01

    Gene expression profiles are used to recognize patient samples for cancer diagnosis and therapy. Gene selection is crucial to high recognition performance. In usual gene selection methods the genes are considered as independent individuals and the correlation among genes is not used efficiently. In this description, a co-expression modules based gene selection method for cancer recognition is proposed. First, in the cancer dataset a weighted correlation network is constructed according to the correlation between each pair of genes, different modules from this network are identified and the significant modules are selected for following exploration. Second, based on these informative modules information gain is applied to selecting the feature genes for cancer recognition. Then using LOOCV, the experiments with different classification algorithms are conducted and the results show that the proposed method makes better classification accuracy than traditional gene selection methods. At last, via gene ontology enrichment analysis the biological significance of the co-expressed genes in specific modules was verified. PMID:24440175

  9. Construction and application of a co-expression network in Mycobacterium tuberculosis.

    PubMed

    Jiang, Jun; Sun, Xian; Wu, Wei; Li, Li; Wu, Hai; Zhang, Lu; Yu, Guohua; Li, Yao

    2016-01-01

    Because of its high pathogenicity and infectivity, tuberculosis is a serious threat to human health. Some information about the functions of the genes in Mycobacterium tuberculosis genome was currently available, but it was not enough to explore transcriptional regulatory mechanisms. Here, we applied the WGCNA (Weighted Gene Correlation Network Analysis) algorithm to mine pooled microarray datasets for the M. tuberculosis H37Rv strain. We constructed a co-expression network that was subdivided into 78 co-expression gene modules. The different response to two kinds of vitro models (a constant 0.2% oxygen hypoxia model and a Wayne model) were explained based on these modules. We identified potential transcription factors based on high Pearson's correlation coefficients between the modules and genes. Three modules that may be associated with hypoxic stimulation were identified, and their potential transcription factors were predicted. In the validation experiment, we determined the expression levels of genes in the modules under hypoxic condition and under overexpression of potential transcription factors (Rv0081, furA (Rv1909c), Rv0324, Rv3334, and Rv3833). The experimental results showed that the three identified modules related to hypoxia and that the overexpression of transcription factors could significantly change the expression levels of genes in the corresponding modules. PMID:27328747

  10. A contribution to the study of plant development evolution based on gene co-expression networks

    PubMed Central

    Romero-Campero, Francisco J.; Lucas-Reina, Eva; Said, Fatima E.; Romero, José M.; Valverde, Federico

    2013-01-01

    Phototrophic eukaryotes are among the most successful organisms on Earth due to their unparalleled efficiency at capturing light energy and fixing carbon dioxide to produce organic molecules. A conserved and efficient network of light-dependent regulatory modules could be at the bases of this success. This regulatory system conferred early advantages to phototrophic eukaryotes that allowed for specialization, complex developmental processes and modern plant characteristics. We have studied light-dependent gene regulatory modules from algae to plants employing integrative-omics approaches based on gene co-expression networks. Our study reveals some remarkably conserved ways in which eukaryotic phototrophs deal with day length and light signaling. Here we describe how a family of Arabidopsis transcription factors involved in photoperiod response has evolved from a single algal gene according to the innovation, amplification and divergence theory of gene evolution by duplication. These modifications of the gene co-expression networks from the ancient unicellular green algae Chlamydomonas reinhardtii to the modern brassica Arabidopsis thaliana may hint on the evolution and specialization of plants and other organisms. PMID:23935602

  11. Uncovering the liver's role in immunity through RNA co-expression networks.

    PubMed

    Harrall, Kylie K; Kechris, Katerina J; Tabakoff, Boris; Hoffman, Paula L; Hines, Lisa M; Tsukamoto, Hidekazu; Pravenec, Michal; Printz, Morton; Saba, Laura M

    2016-10-01

    Gene co-expression analysis has proven to be a powerful tool for ascertaining the organization of gene products into networks that are important for organ function. An organ, such as the liver, engages in a multitude of functions important for the survival of humans, rats, and other animals; these liver functions include energy metabolism, metabolism of xenobiotics, immune system function, and hormonal homeostasis. With the availability of organ-specific transcriptomes, we can now examine the role of RNA transcripts (both protein-coding and non-coding) in these functions. A systems genetic approach for identifying and characterizing liver gene networks within a recombinant inbred panel of rats was used to identify genetically regulated transcriptional networks (modules). For these modules, biological consensus was found between functional enrichment analysis and publicly available phenotypic quantitative trait loci (QTL). In particular, the biological function of two liver modules could be linked to immune response. The eigengene QTLs for these co-expression modules were located at genomic regions coincident with highly significant phenotypic QTLs; these phenotypes were related to rheumatoid arthritis, food preference, and basal corticosterone levels in rats. Our analysis illustrates that genetically and biologically driven RNA-based networks, such as the ones identified as part of this research, provide insight into the genetic influences on organ functions. These networks can pinpoint phenotypes that manifest through the interaction of many organs/tissues and can identify unannotated or under-annotated RNA transcripts that play a role in these phenotypes. PMID:27401171

  12. Co-expression networks in generation of induced pluripotent stem cells.

    PubMed

    Paul, Sharan; Pflieger, Lance; Dansithong, Warunee; Figueroa, Karla P; Gao, Fuying; Coppola, Giovanni; Pulst, Stefan M

    2016-01-01

    We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation. PMID:26892236

  13. Construction and application of a co-expression network in Mycobacterium tuberculosis

    PubMed Central

    Jiang, Jun; Sun, Xian; Wu, Wei; Li, Li; Wu, Hai; Zhang, Lu; Yu, Guohua; Li, Yao

    2016-01-01

    Because of its high pathogenicity and infectivity, tuberculosis is a serious threat to human health. Some information about the functions of the genes in Mycobacterium tuberculosis genome was currently available, but it was not enough to explore transcriptional regulatory mechanisms. Here, we applied the WGCNA (Weighted Gene Correlation Network Analysis) algorithm to mine pooled microarray datasets for the M. tuberculosis H37Rv strain. We constructed a co-expression network that was subdivided into 78 co-expression gene modules. The different response to two kinds of vitro models (a constant 0.2% oxygen hypoxia model and a Wayne model) were explained based on these modules. We identified potential transcription factors based on high Pearson’s correlation coefficients between the modules and genes. Three modules that may be associated with hypoxic stimulation were identified, and their potential transcription factors were predicted. In the validation experiment, we determined the expression levels of genes in the modules under hypoxic condition and under overexpression of potential transcription factors (Rv0081, furA (Rv1909c), Rv0324, Rv3334, and Rv3833). The experimental results showed that the three identified modules related to hypoxia and that the overexpression of transcription factors could significantly change the expression levels of genes in the corresponding modules. PMID:27328747

  14. AuPairWise: A Method to Estimate RNA-Seq Replicability through Co-expression

    PubMed Central

    Ballouz, Sara; Gillis, Jesse

    2016-01-01

    In addition to detecting novel transcripts and higher dynamic range, a principal claim for RNA-sequencing has been greater replicability, typically measured in sample-sample correlations of gene expression levels. Through a re-analysis of ENCODE data, we show that replicability of transcript abundances will provide misleading estimates of the replicability of conditional variation in transcript abundances (i.e., most expression experiments). Heuristics which implicitly address this problem have emerged in quality control measures to obtain ‘good’ differential expression results. However, these methods involve strict filters such as discarding low expressing genes or using technical replicates to remove discordant transcripts, and are costly or simply ad hoc. As an alternative, we model gene-level replicability of differential activity using co-expressing genes. We find that sets of housekeeping interactions provide a sensitive means of estimating the replicability of expression changes, where the co-expressing pair can be regarded as pseudo-replicates of one another. We model the effects of noise that perturbs a gene’s expression within its usual distribution of values and show that perturbing expression by only 5% within that range is readily detectable (AUROC~0.73). We have made our method available as a set of easily implemented R scripts. PMID:27082953

  15. Sharing and Specificity of Co-expression Networks across 35 Human Tissues

    PubMed Central

    Pierson, Emma; Koller, Daphne; Battle, Alexis; Mostafavi, Sara

    2015-01-01

    To understand the regulation of tissue-specific gene expression, the GTEx Consortium generated RNA-seq expression data for more than thirty distinct human tissues. This data provides an opportunity for deriving shared and tissue specific gene regulatory networks on the basis of co-expression between genes. However, a small number of samples are available for a majority of the tissues, and therefore statistical inference of networks in this setting is highly underpowered. To address this problem, we infer tissue-specific gene co-expression networks for 35 tissues in the GTEx dataset using a novel algorithm, GNAT, that uses a hierarchy of tissues to share data between related tissues. We show that this transfer learning approach increases the accuracy with which networks are learned. Analysis of these networks reveals that tissue-specific transcription factors are hubs that preferentially connect to genes with tissue specific functions. Additionally, we observe that genes with tissue-specific functions lie at the peripheries of our networks. We identify numerous modules enriched for Gene Ontology functions, and show that modules conserved across tissues are especially likely to have functions common to all tissues, while modules that are upregulated in a particular tissue are often instrumental to tissue-specific function. Finally, we provide a web tool, available at mostafavilab.stat.ubc.ca/GNAT, which allows exploration of gene function and regulation in a tissue-specific manner. PMID:25970446

  16. Co-expression networks in generation of induced pluripotent stem cells

    PubMed Central

    Paul, Sharan; Pflieger, Lance; Dansithong, Warunee; Figueroa, Karla P.; Gao, Fuying; Coppola, Giovanni; Pulst, Stefan M.

    2016-01-01

    ABSTRACT We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation. PMID:26892236

  17. Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y

    PubMed Central

    Catino, Stefania; Paciello, Fabiola; Miceli, Fiorella; Rolesi, Rolando; Troiani, Diana; Calabrese, Vittorio; Santangelo, Rosaria; Mancuso, Cesare

    2016-01-01

    Over the past years, several lines of evidence have pointed out the efficacy of ferulic acid (FA) in counteracting oxidative stress elicited by β-amyloid or free radical initiators, based on the ability of this natural antioxidant to up-regulate the heme oxygenase-1 (HO-1) and biliverdin reductase (BVR) system. However, scarce results can be found in literature regarding the cytoprotective effects of FA in case of damage caused by neurotoxicants. The aim of this work is to investigate the mechanisms through which FA exerts neuroprotection in SH-SY5Y neuroblastoma cells exposed to the neurotoxin trimethyltin (TMT). FA (1–10 μM for 6 h) dose-dependently increased both basal and TMT (10 μM for 24 h)-induced HO-1 expression in SH-SY5Y cells by fostering the nuclear translocation of the transcriptional activator Nrf2. In particular, the co-treatment of FA (10 μM) with TMT was also responsible for the nuclear translocation of HO-1 in an attempt to further increase cell stress response in SH-SY5Y cells. In addition to HO-1, FA (1–10 μM for 6 h) dose-dependently increased the basal expression of BVR. The antioxidant and neuroprotective features of FA, through the increase of HO activity, were supported by the evidence that FA inhibited TMT (10 μM)-induced lipid peroxidation (evaluated by detecting 4-hydroxy-nonenal) and DNA fragmentation in SH-SY5Y cells and that this antioxidant effect was reversed by the HO inhibitor Zinc-protoporphyrin-IX (5 μM). Among the by-products of the HO/BVR system, carbon monoxide (CORM-2, 50 nM) and bilirubin (BR, 50 nM) significantly inhibited TMT-induced superoxide anion formation in SH-SY5Y cells. All together, these results corroborate the neuroprotective effect of FA through the up-regulation of the HO-1/BVR system, via carbon monoxide and BR formation, and provide the first evidence on the role of HO-1/Nrf2 axis in FA-related enhancement of cell stress response in human neurons. PMID:26779023

  18. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies.

    PubMed

    Marquart, Gregory D; Tabor, Kathryn M; Brown, Mary; Strykowski, Jennifer L; Varshney, Gaurav K; LaFave, Matthew C; Mueller, Thomas; Burgess, Shawn M; Higashijima, Shin-Ichi; Burgess, Harold A

    2015-01-01

    Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3' untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish. PMID:26635538

  19. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies

    PubMed Central

    Marquart, Gregory D.; Tabor, Kathryn M.; Brown, Mary; Strykowski, Jennifer L.; Varshney, Gaurav K.; LaFave, Matthew C.; Mueller, Thomas; Burgess, Shawn M.; Higashijima, Shin-ichi; Burgess, Harold A.

    2015-01-01

    Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3′ untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish. PMID:26635538

  20. Identification of Common Regulators of Genes in Co-Expression Networks Affecting Muscle and Meat Properties

    PubMed Central

    Ponsuksili, Siriluck; Siengdee, Puntita; Du, Yang; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus

    2015-01-01

    Understanding the genetic contributions behind skeletal muscle composition and metabolism is of great interest in medicine and agriculture. Attempts to dissect these complex traits combine genome-wide genotyping, expression data analyses and network analyses. Weighted gene co-expression network analysis (WGCNA) groups genes into modules based on patterns of co-expression, which can be linked to phenotypes by correlation analysis of trait values and the module eigengenes, i.e. the first principal component of a given module. Network hub genes and regulators of the genes in the modules are likely to play an important role in the emergence of respective traits. In order to detect common regulators of genes in modules showing association with meat quality traits, we identified eQTL for each of these genes, including the highly connected hub genes. Additionally, the module eigengene values were used for association analyses in order to derive a joint eQTL for the respective module. Thereby major sites of orchestrated regulation of genes within trait-associated modules were detected as hotspots of eQTL of many genes of a module and of its eigengene. These sites harbor likely common regulators of genes in the modules. We exemplarily showed the consistent impact of candidate common regulators on the expression of members of respective modules by RNAi knockdown experiments. In fact, Cxcr7 was identified and validated as a regulator of genes in a module, which is involved in the function of defense response in muscle cells. Zfp36l2 was confirmed as a regulator of genes of a module related to cell death or apoptosis pathways. The integration of eQTL in module networks enabled to interpret the differentially-regulated genes from a systems perspective. By integrating genome-wide genomic and transcriptomic data, employing co-expression and eQTL analyses, the study revealed likely regulators that are involved in the fine-tuning and synchronization of genes with trait

  1. Identification of key genes for laryngeal squamous cell carcinoma using weighted co-expression network analysis

    PubMed Central

    LI, XIAO-TIAN

    2016-01-01

    Laryngeal squamous cell carcinoma (LSCC) is the most common malignant tumor in the head and neck, and can seriously affect the daily life of patients. To study the mechanisms of LSCC, the microarray of GSE51958 was analyzed in the present study. GSE51958 was downloaded from Gene Expression Omnibus, and included a collection of LSCC tissue samples and matched adjacent non-cancerous tissue samples from 10 patients. Differentially-expressed genes (DEGs) were identified using limma package. Next, a weighted co-expression network was constructed for the DEGs by WGCNA package in R. Modules of the weighted co-expression network were obtained through constructing a hierarchical clustering tree using the hybrid dynamic shear tree method. Using the clusterProfiler package, the potential functions of DEGs in the modules correlated with LSCC were predicted by pathway enrichment analysis. In total, 959 DEGs were screened from the LSCC samples compared with the adjacent non-cancerous samples, including 553 upregulated and 406 downregulated genes. The appointed black, brown, gray, pink and yellow modules were screened for the DEGs in the weighted co-expression network. For the DEGs in the brown and yellow modules, the enriched pathways were cytokine-cytokine receptor interaction and metabolic pathways, respectively. The DEGs in the pink module were involved in the majority of pathways. With high connectivity degrees in the pink module, TPX2, microtubule-associated (TPX2; degree, 25), minichromosome maintenance complex component 2 (MCM2; degree, 25), ubiquitin-like with PHD and ring finger domains 1 (UHRF1; degree, 22), cyclin-dependent kinase 2 (CDK2; degree, 20) and protein regulator of cytokinesis 1 (PRC1; degree, 20) may be involved in LSCC. Overall, In conclusion, from the integrated bioinformatics analysis of genes that may be associated with LSCC, 959 DEGs were obtained from LSCC samples compared with adjacent non-cancerous samples, and TPX2, MCM2, UHRF1, CDK2 and PRC1 were

  2. Identification of common regulators of genes in co-expression networks affecting muscle and meat properties.

    PubMed

    Ponsuksili, Siriluck; Siengdee, Puntita; Du, Yang; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus

    2015-01-01

    Understanding the genetic contributions behind skeletal muscle composition and metabolism is of great interest in medicine and agriculture. Attempts to dissect these complex traits combine genome-wide genotyping, expression data analyses and network analyses. Weighted gene co-expression network analysis (WGCNA) groups genes into modules based on patterns of co-expression, which can be linked to phenotypes by correlation analysis of trait values and the module eigengenes, i.e. the first principal component of a given module. Network hub genes and regulators of the genes in the modules are likely to play an important role in the emergence of respective traits. In order to detect common regulators of genes in modules showing association with meat quality traits, we identified eQTL for each of these genes, including the highly connected hub genes. Additionally, the module eigengene values were used for association analyses in order to derive a joint eQTL for the respective module. Thereby major sites of orchestrated regulation of genes within trait-associated modules were detected as hotspots of eQTL of many genes of a module and of its eigengene. These sites harbor likely common regulators of genes in the modules. We exemplarily showed the consistent impact of candidate common regulators on the expression of members of respective modules by RNAi knockdown experiments. In fact, Cxcr7 was identified and validated as a regulator of genes in a module, which is involved in the function of defense response in muscle cells. Zfp36l2 was confirmed as a regulator of genes of a module related to cell death or apoptosis pathways. The integration of eQTL in module networks enabled to interpret the differentially-regulated genes from a systems perspective. By integrating genome-wide genomic and transcriptomic data, employing co-expression and eQTL analyses, the study revealed likely regulators that are involved in the fine-tuning and synchronization of genes with trait

  3. Protein-protein interaction and gene co-expression maps of ARFs and Aux/IAAs in Arabidopsis

    PubMed Central

    Piya, Sarbottam; Shrestha, Sandesh K.; Binder, Brad; Stewart, C. Neal; Hewezi, Tarek

    2014-01-01

    The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis thaliana, Auxin/indole-3-acetic acid (Aux/IAA) proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins are related to their unique biological functions. The objective of this study was to generate the Aux/IAA-ARF protein-protein interaction map using full length sequences and locate the interacting protein pairs to specific gene co-expression networks in order to define tissue-specific responses of the Aux/IAA-ARF interactome. Pairwise interactions between 19 ARFs and 29 Aux/IAAs resulted in the identification of 213 specific interactions of which 79 interactions were previously unknown. The incorporation of co-expression profiles with protein-protein interaction data revealed a strong correlation of gene co-expression for 70% of the ARF-Aux/IAA interacting pairs in at least one tissue/organ, indicative of the biological significance of these interactions. Importantly, ARF4-8 and 19, which were found to interact with almost all Aux-Aux/IAA showed broad co-expression relationships with Aux/IAA genes, thus, formed the central hubs of the co-expression network. Our analyses provide new insights into the biological significance of ARF-Aux/IAA associations in the morphogenesis and development of various plant tissues and organs. PMID:25566309

  4. Computational gene expression profiling under salt stress reveals patterns of co-expression.

    PubMed

    Sanchita; Sharma, Ashok

    2016-03-01

    Plants respond differently to environmental conditions. Among various abiotic stresses, salt stress is a condition where excess salt in soil causes inhibition of plant growth. To understand the response of plants to the stress conditions, identification of the responsible genes is required. Clustering is a data mining technique used to group the genes with similar expression. The genes of a cluster show similar expression and function. We applied clustering algorithms on gene expression data of Solanum tuberosum showing differential expression in Capsicum annuum under salt stress. The clusters, which were common in multiple algorithms were taken further for analysis. Principal component analysis (PCA) further validated the findings of other cluster algorithms by visualizing their clusters in three-dimensional space. Functional annotation results revealed that most of the genes were involved in stress related responses. Our findings suggest that these algorithms may be helpful in the prediction of the function of co-expressed genes. PMID:26981411

  5. Co-expression of Endoxylanase and Endoglucanase in Scheffersomyces stipitis and Its Application in Ethanol Production.

    PubMed

    Puseenam, Aekkachai; Tanapongpipat, Sutipa; Roongsawang, Niran

    2015-12-01

    Scheffersomyces stipitis strain BCC15191 is considered as a biotechnologically valuable yeast for its ability to ferment glucose and xylose, the main sugar components in plant biomass, to ethanol. However, the wild strain lacks of endogenous cellulases and hemicellulases that limited biomass utilization. In order to improve biomass degrading ability of S. stipitis BCC15191, new integrative plasmids harboring constitutive TEF1 promoter and codon-optimized zeocin or hygromycin antibiotic resistance genes were developed. Aspergillus niger endoxylanase and Aspergillus aculeatus endoglucanase activities were demonstrated in transformant cells expressing codon-optimized genes. S. stipitis co-expressing endoxylanase and endoglucanase was able to grow in medium containing xylan and β-glucan as carbon sources and directly produced ethanol with yields of 2.7 g/L. It could also use pretreated corncob as a carbon source for ethanol production. These results suggested that recombinant S. stipilis is possible for consolidated bioprocessing of biomass. PMID:26378014

  6. Co-expression network analysis of Down's syndrome based on microarray data

    PubMed Central

    Zhao, Jianping; Zhang, Zhengguo; Ren, Shumin; Zong, Yanan; Kong, Xiangdong

    2016-01-01

    Down's syndrome (DS) is a type of chromosome disease. The present study aimed to explore the underlying molecular mechanisms of DS. GSE5390 microarray data downloaded from the gene expression omnibus database was used to identify differentially expressed genes (DEGs) in DS. Pathway enrichment analysis of the DEGs was performed, followed by co-expression network construction. Significant differential modules were mined by mutual information, followed by functional analysis. The accuracy of sample classification for the significant differential modules of DEGs was evaluated by leave-one-out cross-validation. A total of 997 DEGs, including 638 upregulated and 359 downregulated genes, were identified. Upregulated DEGs were enriched in 15 pathways, such as cell adhesion molecules, whereas downregulated DEGs were enriched in maturity onset diabetes of the young. Three significant differential modules with the highest discriminative scores (mutual information>0.35) were selected from a co-expression network. The classification accuracy of GSE16677 expression profile samples was 54.55% and 72.73% when characterized by 12 DEGs and 3 significant differential modules, respectively. Genes in significant differential modules were significantly enriched in 5 functions, including the endoplasmic reticulum (P=0.018) and regulation of apoptosis (P=0.061). The identified DEGs, in particular the 12 DEGs in the significant differential modules, such as B-cell lymphoma 2-associated transcription factor 1, heat shock protein 90 kDa beta member 1, UBX domain-containing protein 2 and transmembrane protein 50B, may serve important roles in the pathogenesis of DS. PMID:27588071

  7. Relating specific connexin co-expression ratio to connexon composition and gap junction function.

    PubMed

    Desplantez, T; Grikscheit, K; Thomas, N M; Peters, N S; Severs, N J; Dupont, E

    2015-12-01

    Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood. This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45. Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction. These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart. PMID:26550940

  8. Energy transfer between fusion biliproteins co-expressed with phycobiliprotein in Escherichia coli.

    PubMed

    Ma, Qiong; Zhou, Nan; Zhou, Ming

    2016-10-01

    In cyanobacteria, phycobiliproteins (PBS) show excellent energy transfer among the chromophores absorbing over most of the visible. The energy transfers are used to study phycobilisome assembly and bioimaging. Using All4261GAF2(C81L) as energy donor, ApcE(1-240/Δ87-130) as energy acceptor, we co-expressed fusion protein ApcE(1-240/Δ87-130)::All4261GAF2(C81L) with phycobiliprotein in Escherichia Coli and studied the energy transfer between two protein domains. With N-terminal His6 tag, ApcE(1-240/Δ87-130)::All4261GAF2(C81L) cannot be purified by nickel-affinity column. We added six histidines in the C-terminal of ApcE(1-240/Δ87-130)::All4261GAF2(C81L) and co-expressed it with phycobiliprotein. ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was purified successfully and only singly chromophorylated at All4261GAF2(C81L)His6 domain. The singly chromophorylate ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was incubated with fresh PCB and the doubly chromophorylated PCB-ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was obtained. The double chromophored fusion protein absorbed light in the range of 615-660 nm, and fluoresced only at 668 nm. Photochemistry analysis showed that excitation energy transfer from the short-wavelength absorbing at All4261GAF2(C81L) domain was achieved successfully to the long-wavelength absorbing at the ApcE(1-240/Δ87-130) domain. PMID:27260968

  9. CD90 and CD24 Co-Expression Is Associated with Pancreatic Intraepithelial Neoplasias

    PubMed Central

    Pei, Xiucong; Zhu, Jianhui; Yang, Rui; Tan, Zhijing; An, Mingrui; Shi, Jiaqi; Lubman, David M.

    2016-01-01

    Thy-1 (CD90) has been shown to be a potential marker for several different types of cancer. However, reports on CD90 expression in pancreatic intraepithelial neoplasia (PanIN) lesions are still limited where PanINs are the most important precursor lesion of pancreatic ductal adenocarcinoma (PDAC). Herein, we investigate candidate markers for PanIN lesions by examining the distribution and trend of CD90 and CD24 expression as well as their co-expression in various stages of PanINs. Thirty cases of PanINs, which were confirmed histopathologically and clinically, were used to evaluate protein expression of CD90 and CD24 by immunofluoresence double staining. CD90 was found to be mainly expressed in stroma around lesion ducts while not observed in acini and islets in PanINs. CD90 also showed increased expression in PanIN III compared to PanIN III. CD24 was mainly present in the cytoplasm and membrane of pancreatic ductal epithelia, especially in the apical epithelium of the duct. CD24 had higher expression in PanIN III compared with PanIN IIIIII or PanIN III. CD90 was expressed around CD24 sites, but there was little overlap between cells that expressed each of these proteins. A correlation analysis showed that these two proteins have a moderate relationship with PanIN stages respectively. These results suggest that co-expression of CD90 and CD24 may have an important role in the development and progression of PanINs, which is also conducive to early detection and treatment of PDAC. PMID:27332878

  10. Differential co-expression analysis of rheumatoid arthritis with microarray data.

    PubMed

    Wang, Kunpeng; Zhao, Liqiang; Liu, Xuefeng; Hao, Zhenyong; Zhou, Yong; Yang, Chuandong; Li, Hongqiang

    2014-11-01

    The aim of the present study was to investigate the underlying molecular mechanisms of rheumatoid arthritis (RA) using microarray expression profiles from osteoarthritis and RA patients, to improve diagnosis and treatment strategies for the condition. The gene expression profile of GSE27390 was downloaded from Gene Expression Omnibus, including 19 samples from patients with RA (n=9) or osteoarthritis (n=10). Firstly, the differentially expressed genes (DEGs) were obtained with the thresholds of |logFC|>1.0 and P<0.05, using the t‑test method in LIMMA package. Then, differentially co-expressed genes (DCGs) and differentially co-expressed links (DCLs) were screened with q<0.25 by the differential coexpression analysis and differential regulation analysis of gene expression microarray data package. Secondly, pathway enrichment analysis for DCGs was performed by the Database for Annotation, Visualization and Integrated Discovery and the DCLs associated with RA were selected by comparing the obtained DCLs with known transcription factor (TF)-targets in the TRANSFAC database. Finally, the obtained TFs were mapped to the known TF-targets to construct the network using cytoscape software. A total of 1755 DEGs, 457 DCGs and 101988 DCLs were achieved and there were 20 TFs in the obtained six TF-target relations (STAT3-TNF, PBX1‑PLAU, SOCS3-STAT3, GATA1-ETS2, ETS1-ICAM4 and CEBPE‑GATA1) and 457 DCGs. A number of TF-target relations in the constructed network were not within DCLs when the TF and target gene were DCGs. The identified TFs may have an important role in the pathogenesis of RA and have the potential to be used as biomarkers for the development of novel diagnostic and therapeutic strategies for RA. PMID:25118911

  11. The Structure of a Gene Co-Expression Network Reveals Biological Functions Underlying eQTLs

    PubMed Central

    Villa-Vialaneix, Nathalie; Liaubet, Laurence; Laurent, Thibault; Cherel, Pierre; Gamot, Adrien; SanCristobal, Magali

    2013-01-01

    What are the commonalities between genes, whose expression level is partially controlled by eQTL, especially with regard to biological functions? Moreover, how are these genes related to a phenotype of interest? These issues are particularly difficult to address when the genome annotation is incomplete, as is the case for mammalian species. Moreover, the direct link between gene expression and a phenotype of interest may be weak, and thus difficult to handle. In this framework, the use of a co-expression network has proven useful: it is a robust approach for modeling a complex system of genetic regulations, and to infer knowledge for yet unknown genes. In this article, a case study was conducted with a mammalian species. It showed that the use of a co-expression network based on partial correlation, combined with a relevant clustering of nodes, leads to an enrichment of biological functions of around 83%. Moreover, the use of a spatial statistics approach allowed us to superimpose additional information related to a phenotype; this lead to highlighting specific genes or gene clusters that are related to the network structure and the phenotype. Three main results are worth noting: first, key genes were highlighted as a potential focus for forthcoming biological experiments; second, a set of biological functions, which support a list of genes under partial eQTL control, was set up by an overview of the global structure of the gene expression network; third, pH was found correlated with gene clusters, and then with related biological functions, as a result of a spatial analysis of the network topology. PMID:23577081

  12. The structure of a gene co-expression network reveals biological functions underlying eQTLs.

    PubMed

    Villa-Vialaneix, Nathalie; Liaubet, Laurence; Laurent, Thibault; Cherel, Pierre; Gamot, Adrien; SanCristobal, Magali

    2013-01-01

    What are the commonalities between genes, whose expression level is partially controlled by eQTL, especially with regard to biological functions? Moreover, how are these genes related to a phenotype of interest? These issues are particularly difficult to address when the genome annotation is incomplete, as is the case for mammalian species. Moreover, the direct link between gene expression and a phenotype of interest may be weak, and thus difficult to handle. In this framework, the use of a co-expression network has proven useful: it is a robust approach for modeling a complex system of genetic regulations, and to infer knowledge for yet unknown genes. In this article, a case study was conducted with a mammalian species. It showed that the use of a co-expression network based on partial correlation, combined with a relevant clustering of nodes, leads to an enrichment of biological functions of around 83%. Moreover, the use of a spatial statistics approach allowed us to superimpose additional information related to a phenotype; this lead to highlighting specific genes or gene clusters that are related to the network structure and the phenotype. Three main results are worth noting: first, key genes were highlighted as a potential focus for forthcoming biological experiments; second, a set of biological functions, which support a list of genes under partial eQTL control, was set up by an overview of the global structure of the gene expression network; third, pH was found correlated with gene clusters, and then with related biological functions, as a result of a spatial analysis of the network topology. PMID:23577081

  13. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence.

    PubMed

    Mamdani, Mohammed; Williamson, Vernell; McMichael, Gowon O; Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; van der Vaart, Andrew D; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S; Miles, Michael F; Dick, Danielle; Riley, Brien P; Dumur, Catherine; Vladimirov, Vladimir I

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  14. Integrating mRNA and miRNA Weighted Gene Co-Expression Networks with eQTLs in the Nucleus Accumbens of Subjects with Alcohol Dependence

    PubMed Central

    Blevins, Tana; Aliev, Fazil; Adkins, Amy; Hack, Laura; Bigdeli, Tim; D. van der Vaart, Andrew; Web, Bradley Todd; Bacanu, Silviu-Alin; Kalsi, Gursharan; Kendler, Kenneth S.; Miles, Michael F.; Dick, Danielle; Riley, Brien P.; Dumur, Catherine; Vladimirov, Vladimir I.

    2015-01-01

    Alcohol consumption is known to lead to gene expression changes in the brain. After performing weighted gene co-expression network analyses (WGCNA) on genome-wide mRNA and microRNA (miRNA) expression in Nucleus Accumbens (NAc) of subjects with alcohol dependence (AD; N = 18) and of matched controls (N = 18), six mRNA and three miRNA modules significantly correlated with AD were identified (Bonferoni-adj. p≤ 0.05). Cell-type-specific transcriptome analyses revealed two of the mRNA modules to be enriched for neuronal specific marker genes and downregulated in AD, whereas the remaining four mRNA modules were enriched for astrocyte and microglial specific marker genes and upregulated in AD. Gene set enrichment analysis demonstrated that neuronal specific modules were enriched for genes involved in oxidative phosphorylation, mitochondrial dysfunction and MAPK signaling. Glial-specific modules were predominantly enriched for genes involved in processes related to immune functions, i.e. cytokine signaling (all adj. p≤ 0.05). In mRNA and miRNA modules, 461 and 25 candidate hub genes were identified, respectively. In contrast to the expected biological functions of miRNAs, correlation analyses between mRNA and miRNA hub genes revealed a higher number of positive than negative correlations (χ2 test p≤ 0.0001). Integration of hub gene expression with genome-wide genotypic data resulted in 591 mRNA cis-eQTLs and 62 miRNA cis-eQTLs. mRNA cis-eQTLs were significantly enriched for AD diagnosis and AD symptom counts (adj. p = 0.014 and p = 0.024, respectively) in AD GWAS signals in a large, independent genetic sample from the Collaborative Study on Genetics of Alcohol (COGA). In conclusion, our study identified putative gene network hubs coordinating mRNA and miRNA co-expression changes in the NAc of AD subjects, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD. PMID:26381263

  15. Am80 induces neuronal differentiation via increased tropomyosin-related kinase B expression in a human neuroblastoma SH-SY5Y cell line.

    PubMed

    Shiohira, Hideo; Kitaoka, Akira; Enjoji, Munechika; Uno, Tsukasa; Nakashima, Manabu

    2012-01-01

    Am80, a synthetic retinoid, has been used in differentiation therapy for acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA) as one of natural retinoid has been also used to treat APL. ATRA treatment causes neuronal differentiation by inducing tropomyosin-related kinase B (TrkB) expression and increasing the sensitivity to brain-derived neurotrophic factor (BDNF), a TrkB ligand. In the present study, we investigated the effects of Am80 on neuronal differentiation, BDNF sensitivity and TrkB expression in human neuroblastoma SH-SY5Y cells. Treatment with Am80 induced morphological differentiation of neurite outgrowth and increased the expression of GAP43 mRNA, a neuronal differentiation marker. Additionally, TrkB protein was also increased, and exogenous BDNF stimulation after treatment with Am80 induced greater neurite outgrowth than without BDNF treatment. These results suggest that Am80 induced neuronal differentiation by increasing TrkB expression and BDNF sensitivity. PMID:23124249

  16. Major histocompatibility complex class I expression on neurons in subacute sclerosing panencephalitis and experimental subacute measles encephalitis

    SciTech Connect

    Gogate, N.; Yamabe, Toshio; Verma, L.; Dhib-Jalbut, S.

    1996-04-01

    Lack of major histocompatibility class I antigens on neurons has been implicated as a possible mechanism for viral persistence in the brain since these antigens are required for cytotoxic T-lymphocyte recognition of infected cells. In subacute sclerosing panencephalitis (SSPE), measles virus (MV) persists in neurons, resulting in a fatal chronic infection. MHC class I mRNA expression was examined in formalin-fixed brain tissue from 6 SSPE patients by in situ hybridization. In addition MHC class I protein expression in MV-infected neurons was examined in experimental Subacute Measles Encephalitis (SME) by double immunohistochemistry. MHC class I mRNA expression was found to be upregulated in SSPE tissues studied, and in 5 out of 6 cases the expression was definitively seen on neurons. The percentage of neurons expressing MHC class I mRNA ranged between 20 to 84% in infected areas. There was no correlation between the degree of infection and expression of MHC class I molecules on neurons. Importantly, the number of neurons co-expressing MHC class I and MV antigens was markedly low, varying between 2 to 8%. Similar results were obtained in SME where 20 to 30% of the neurons expressed MHC class I but < 8% co-expressed MHC class I and MV antigens. Perivascular infiltrating cells in the infected regions in SME expressed IFN{gamma} immunoreactivity. The results suggest that MV may not be directly involved in the induction of MHC class I on neurons and that cytokines such as IFN{gamma} may play an important role. Furthermore, the paucity of neurons co-expressing MHC class I and MV antigens in SSPE and SME suggests that such cells are either rapidly cleared by cytotoxic T lymphocytes (CTL), or, alternatively, lack of co-expression of MHC class I on MV infected neurons favors MV persistence in these cells by escaping CTL recognition. 33 refs., 3 figs., 3 tabs.

  17. Discovery of core biotic stress responsive genes in Arabidopsis by weighted gene co-expression network analysis.

    PubMed

    Amrine, Katherine C H; Blanco-Ulate, Barbara; Cantu, Dario

    2015-01-01

    Intricate signal networks and transcriptional regulators translate the recognition of pathogens into defense responses. In this study, we carried out a gene co-expression analysis of all currently publicly available microarray data, which were generated in experiments that studied the interaction of the model plant Arabidopsis thaliana with microbial pathogens. This work was conducted to identify (i) modules of functionally related co-expressed genes that are differentially expressed in response to multiple biotic stresses, and (ii) hub genes that may function as core regulators of disease responses. Using Weighted Gene Co-expression Network Analysis (WGCNA) we constructed an undirected network leveraging a rich curated expression dataset comprising 272 microarrays that involved microbial infections of Arabidopsis plants with a wide array of fungal and bacterial pathogens with biotrophic, hemibiotrophic, and necrotrophic lifestyles. WGCNA produced a network with scale-free and small-world properties composed of 205 distinct clusters of co-expressed genes. Modules of functionally related co-expressed genes that are differentially regulated in response to multiple pathogens were identified by integrating differential gene expression testing with functional enrichment analyses of gene ontology terms, known disease associated genes, transcriptional regulators, and cis-regulatory elements. The significance of functional enrichments was validated by comparisons with randomly generated networks. Network topology was then analyzed to identify intra- and inter-modular gene hubs. Based on high connectivity, and centrality in meta-modules that are clearly enriched in defense responses, we propose a list of 66 target genes for reverse genetic experiments to further dissect the Arabidopsis immune system. Our results show that statistical-based data trimming prior to network analysis allows the integration of expression datasets generated by different groups, under different

  18. Discovery of Core Biotic Stress Responsive Genes in Arabidopsis by Weighted Gene Co-Expression Network Analysis

    PubMed Central

    Amrine, Katherine C. H.; Blanco-Ulate, Barbara; Cantu, Dario

    2015-01-01

    Intricate signal networks and transcriptional regulators translate the recognition of pathogens into defense responses. In this study, we carried out a gene co-expression analysis of all currently publicly available microarray data, which were generated in experiments that studied the interaction of the model plant Arabidopsis thaliana with microbial pathogens. This work was conducted to identify (i) modules of functionally related co-expressed genes that are differentially expressed in response to multiple biotic stresses, and (ii) hub genes that may function as core regulators of disease responses. Using Weighted Gene Co-expression Network Analysis (WGCNA) we constructed an undirected network leveraging a rich curated expression dataset comprising 272 microarrays that involved microbial infections of Arabidopsis plants with a wide array of fungal and bacterial pathogens with biotrophic, hemibiotrophic, and necrotrophic lifestyles. WGCNA produced a network with scale-free and small-world properties composed of 205 distinct clusters of co-expressed genes. Modules of functionally related co-expressed genes that are differentially regulated in response to multiple pathogens were identified by integrating differential gene expression testing with functional enrichment analyses of gene ontology terms, known disease associated genes, transcriptional regulators, and cis-regulatory elements. The significance of functional enrichments was validated by comparisons with randomly generated networks. Network topology was then analyzed to identify intra- and inter-modular gene hubs. Based on high connectivity, and centrality in meta-modules that are clearly enriched in defense responses, we propose a list of 66 target genes for reverse genetic experiments to further dissect the Arabidopsis immune system. Our results show that statistical-based data trimming prior to network analysis allows the integration of expression datasets generated by different groups, under different

  19. ComPlEx: conservation and divergence of co-expression networks in A. thaliana, Populus and O. sativa

    PubMed Central

    2014-01-01

    Background Divergence in gene regulation has emerged as a key mechanism underlying species differentiation. Comparative analysis of co-expression networks across species can reveal conservation and divergence in the regulation of genes. Results We inferred co-expression networks of A. thaliana, Populus spp. and O. sativa using state-of-the-art methods based on mutual information and context likelihood of relatedness, and conducted a comprehensive comparison of these networks across a range of co-expression thresholds. In addition to quantifying gene-gene link and network neighbourhood conservation, we also applied recent advancements in network analysis to do cross-species comparisons of network properties such as scale free characteristics and gene centrality as well as network motifs. We found that in all species the networks emerged as scale free only above a certain co-expression threshold, and that the high-centrality genes upholding this organization tended to be conserved. Network motifs, in particular the feed-forward loop, were found to be significantly enriched in specific functional subnetworks but where much less conserved across species than gene centrality. Although individual gene-gene co-expression had massively diverged, up to ~80% of the genes still had a significantly conserved network neighbourhood. For genes with multiple predicted orthologs, about half had one ortholog with conserved regulation and another ortholog with diverged or non-conserved regulation. Furthermore, the most sequence similar ortholog was not the one with the most conserved gene regulation in over half of the cases. Conclusions We have provided a comprehensive analysis of gene regulation evolution in plants and built a web tool for Comparative analysis of Plant co-Expression networks (ComPlEx, http://complex.plantgenie.org/). The tool can be particularly useful for identifying the ortholog with the most conserved regulation among several sequence-similar alternatives and

  20. Neuronal polarization.

    PubMed

    Takano, Tetsuya; Xu, Chundi; Funahashi, Yasuhiro; Namba, Takashi; Kaibuchi, Kozo

    2015-06-15

    Neurons are highly polarized cells with structurally and functionally distinct processes called axons and dendrites. This polarization underlies the directional flow of information in the central nervous system, so the establishment and maintenance of neuronal polarization is crucial for correct development and function. Great progress in our understanding of how neurons establish their polarity has been made through the use of cultured hippocampal neurons, while recent technological advances have enabled in vivo analysis of axon specification and elongation. This short review and accompanying poster highlight recent advances in this fascinating field, with an emphasis on the signaling mechanisms underlying axon and dendrite specification in vitro and in vivo. PMID:26081570

  1. Co-expression of laminin β3 and γ2 chains and epigenetic inactivation of laminin α3 chain in gastric cancer.

    PubMed

    Ii, Masanori; Yamamoto, Hiroyuki; Taniguchi, Hiroaki; Adachi, Yasushi; Nakazawa, Mayumi; Ohashi, Hirokazu; Tanuma, Tokuma; Sukawa, Yasutaka; Suzuki, Hiromu; Sasaki, Shigeru; Imai, Kohzoh; Shinomura, Yasuhisa

    2011-09-01

    Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that regulates cell adhesion and migration. Molecular alterations of LM-332 are involved in cancer progression. The aim of this study was to clarify alterations of LM-332 in gastric carcinoma. The expression of LM-332 subunits in 10 gastric carcinoma cell lines was investigated by RT-PCR, Western blotting, and immuno-cytochemical/immunofluorescent analyses. The promoter methylation status of LM-332-encoding genes (LAMA3, LAMB3 and LAMC2) was analyzed by methylation-specific PCR (MSP). The relationship between cell migration and LM-332 expression was assessed by the scratch assay. The expression of LM-332 was analyzed immunohistochemically in 90 gastric cancer tissues. Co-expression of laminin β3 and γ2 chains was often observed in gastric carcinoma cell lines at mRNA and protein levels. In contrast, there was no expression of laminin α3 at either the mRNA or protein levels. Extra-cellular secretion of laminin β3 and γ2 chains was found in 2 of the 10 cell lines. The LAMA3 gene was transcriptionally silenced by methylation of the promoter CpG islands in all of the cell lines, while the LAMB3 and LAMC2 genes were silenced in several cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored expression of the LM-332-encoding genes. Methylation frequency of LAMA3 was higher than those of the LAMB2 and LAMC2 genes in gastric cancer tissues. Migration distances were significantly correlated with cytoplasmic laminin γ2 chain expression. Immunohistochemistry showed frequent co-expression of laminin β3 and γ2 chains in gastric carcinoma cells, which was significantly correlated with depth of invasion and advanced tumor stage. The results suggest that the laminin β3 and γ2 chains accumulate intracellularly and play a role in gastric cancer progression, while epigenetic silencing of the laminin α3 chain may lead to inability to synthesize the basement

  2. Human Mas-Related G Protein-Coupled Receptors-X1 Induce Chemokine Receptor 2 Expression in Rat Dorsal Root Ganglia Neurons and Release of Chemokine Ligand 2 from the Human LAD-2 Mast Cell Line

    PubMed Central

    Solinski, Hans Jürgen; Petermann, Franziska; Rothe, Kathrin; Boekhoff, Ingrid; Gudermann, Thomas; Breit, Andreas

    2013-01-01

    Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain. PMID:23505557

  3. NLRP3 inflammasome activation in macrophage cell lines by prion protein fibrils as the source of IL-1β and neuronal toxicity

    PubMed Central

    Hafner-Bratkovič, Iva; Benčina, Mojca; Fitzgerald, Katherine A.; Golenbock, Douglas; Jerala, Roman

    2012-01-01

    Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, neuronal loss and activation of astrocytes and microglia. Microglia can clear prion plaques but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K+ and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease. PMID:22926439

  4. Parvalbumin+ Neurons and Npas1+ Neurons Are Distinct Neuron Classes in the Mouse External Globus Pallidus

    PubMed Central

    Hernández, Vivian M.; Hegeman, Daniel J.; Cui, Qiaoling; Kelver, Daniel A.; Fiske, Michael P.; Glajch, Kelly E.; Pitt, Jason E.; Huang, Tina Y.; Justice, Nicholas J.

    2015-01-01

    Compelling evidence suggests that pathological activity of the external globus pallidus (GPe), a nucleus in the basal ganglia, contributes to the motor symptoms of a variety of movement disorders such as Parkinson's disease. Recent studies have challenged the idea that the GPe comprises a single, homogenous population of neurons that serves as a simple relay in the indirect pathway. However, we still lack a full understanding of the diversity of the neurons that make up the GPe. Specifically, a more precise classification scheme is needed to better describe the fundamental biology and function of different GPe neuron classes. To this end, we generated a novel multicistronic BAC (bacterial artificial chromosome) transgenic mouse line under the regulatory elements of the Npas1 gene. Using a combinatorial transgenic and immunohistochemical approach, we discovered that parvalbumin-expressing neurons and Npas1-expressing neurons in the GPe represent two nonoverlapping cell classes, amounting to 55% and 27% of the total GPe neuron population, respectively. These two genetically identified cell classes projected primarily to the subthalamic nucleus and to the striatum, respectively. Additionally, parvalbumin-expressing neurons and Npas1-expressing neurons were distinct in their autonomous and driven firing characteristics, their expression of intrinsic ion conductances, and their responsiveness to chronic 6-hydroxydopamine lesion. In summary, our data argue that parvalbumin-expressing neurons and Npas1-expressing neurons are two distinct functional classes of GPe neurons. This work revises our understanding of the GPe, and provides the foundation for future studies of its function and dysfunction. SIGNIFICANCE STATEMENT Until recently, the heterogeneity of the constituent neurons within the external globus pallidus (GPe) was not fully appreciated. We addressed this knowledge gap by discovering two principal GPe neuron classes, which were identified by their nonoverlapping

  5. Co-expression of mouse TMEM63A, TMEM63B and TMEM63C confers hyperosmolarity activated ion currents in HEK293 cells.

    PubMed

    Zhao, Xin; Yan, Xiaojuan; Liu, Yuanhu; Zhang, Ping; Ni, Xin

    2016-06-01

    Osmoreception is essential for systemic osmoregulation, a process to stabilize the tonicity and volume of the extracellular fluid through regulating the ingestive behaviour, sympathetic outflow and renal function. The sensation of osmotic changes by osmoreceptor neurons is mediated by ion channels that detect the change of osmolarity in extracellular fluid. However, the molecular identity of these channels remains mysterious. AtCSC1and OSCA1,two closely related paralogues from Arabidopsis, have been demonstrated to form hyperosmolarity activated ion channels, which makes their mammalian orthologues-the members of TMEM63 proteins, possible candidates for osmoreceptor transduction channel. To test this possibility, we cloned the cDNAs of all the three members of the mouse TMEM63 family, TMEM63A, TMEM63B and TMEM63C from the mRNA from mouse brain. When all of the three subtypes of TMEM63 proteins were co-expressed in HEK293 cells, we recorded membrane currents evoked by hypertonic stimulation in these cells. However, the cells expressing the combinations of any two subtypes of TMEM63 proteins could not exhibit any hyperosmolarity evoked currents. Thus, all the three members of TMEM63 proteins are required to constitute a hyperosmolarity activated ion channel. We propose that the TMEM63 proteins may serve as an osmolarity sensitive ion channel for the osmoreception. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27045885

  6. Modeling the effects of HER/ErbB1-3 co-expression on receptor dimerization and biological response

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.; Resat, Haluk

    2006-06-01

    The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologues viz. HER2-4. This receptor system plays a critical role in cell proliferation and differentiation and receptor over-expression can be associated with poor prognosis in cancers of the epithelium. Here, we examine the effect of co-expressing varying levels of HER1-3 on the receptor dimerization patterns using a detailed kinetic model for ErbB heterodimerization and trafficking. Our results indicate that co-expression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal down-regulation. In addition, simultaneous co-expression of HER1-3 leads to preferential formation of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. Analysis of the parameter dependencies in the model reveals that measurements of HER3 phosphorylation and HER2 internalization ratio may prove to be especially useful for the estimation of critical model parameters. Further, we examined the effect of receptor dimerization patterns on cell phenotype using a simple phenomenological model. Results indicate that co-expression of EGFR with HER2 and HER3 at low to moderate levels may enable cells to match the phenotype of a high HER2 expresser.

  7. Co-expression as a convenient method for the production and purification of core histones in bacteria.

    PubMed

    Anderson, Megan; Huh, Joon H; Ngo, Thien; Lee, Alice; Hernandez, Genaro; Pang, Joy; Perkins, Jennifer; Dutnall, Robert N

    2010-08-01

    Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity. PMID:20347990

  8. Novel role of ZmaNAC36 in co-expression of starch synthetic genes in maize endosperm.

    PubMed

    Zhang, Junjie; Chen, Jiang; Yi, Qiang; Hu, Yufeng; Liu, Hanmei; Liu, Yinghong; Huang, Yubi

    2014-02-01

    Starch is an essential commodity that is widely used as food, feed, fuel and in industry. However, its mechanism of synthesis is not fully understood, especially in terms of the expression and regulation of the starch synthetic genes. It was reported that the starch synthetic genes were co-expressed during maize endosperm development; however, the mechanism of the co-expression was not reported. In this paper, the ZmaNAC36 gene was amplified by homology-based cloning, and its expression vector was constructed for transient expression. The nuclear localization, transcriptional activation and target sites of the ZmaNAC36 protein were identified. The expression profile of ZmaNAC36 showed that it was strongly expressed in the maize endosperm and was co-expressed with most of the starch synthetic genes. Moreover, the expressions of many starch synthesis genes in the endosperm were upregulated when ZmaNAC36 was transiently overexpressed. All our results indicated that NAC36 might be a transcription factor and play a potential role in the co-expression of starch synthetic genes in the maize endosperm. PMID:24235061

  9. Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce.

    PubMed

    Lamara, Mebarek; Raherison, Elie; Lenz, Patrick; Beaulieu, Jean; Bousquet, Jean; MacKay, John

    2016-04-01

    Association studies are widely utilized to analyze complex traits but their ability to disclose genetic architectures is often limited by statistical constraints, and functional insights are usually minimal in nonmodel organisms like forest trees. We developed an approach to integrate association mapping results with co-expression networks. We tested single nucleotide polymorphisms (SNPs) in 2652 candidate genes for statistical associations with wood density, stiffness, microfibril angle and ring width in a population of 1694 white spruce trees (Picea glauca). Associations mapping identified 229-292 genes per wood trait using a statistical significance level of P < 0.05 to maximize discovery. Over-representation of genes associated for nearly all traits was found in a xylem preferential co-expression group developed in independent experiments. A xylem co-expression network was reconstructed with 180 wood associated genes and several known MYB and NAC regulators were identified as network hubs. The network revealed a link between the gene PgNAC8, wood stiffness and microfibril angle, as well as considerable within-season variation for both genetic control of wood traits and gene expression. Trait associations were distributed throughout the network suggesting complex interactions and pleiotropic effects. Our findings indicate that integration of association mapping and co-expression networks enhances our understanding of complex wood traits. PMID:26619072

  10. Co-expression of cystatin inhibitors OCI and OCII in transgenic potato plants alters Colorado potato beetle development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oryzacystatins I and II (OCI and OCII) show potential for controlling pests that utilize cysteine proteinases for protein digestion. To strengthen individual inhibitory range and achieve an additive effect in the overall efficiency of these proteins against pests, both cystatin genes were co-express...

  11. G-NEST: A gene neighborhood scoring tool to identify co-conserved, co-expressed genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous studies, gene neighborhoods--spatial clusters of co-expressed genes in the genome--have been defined using arbitrary rules such as requiring adjacency, a minimum number of genes, a fixed window size, or a minimum expression level. In the current study, we developed a Gene Neighborhood Sc...

  12. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    PubMed Central

    2012-01-01

    Background In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in

  13. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma.

    PubMed

    Song, Qingfeng; Zhao, Chang; Ou, Shengqiu; Meng, Zhibin; Kang, Ping; Fan, Liwei; Qi, Feng; Ma, Yilong

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  14. [Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus].

    PubMed

    Xu, Peng-Wei; Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Liu, Xiao-Yu; Zeng, Xian-Yin; Zhang, Zhi-Qing

    2012-05-01

    The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines. PMID:22764525

  15. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma

    PubMed Central

    SONG, QINGFENG; ZHAO, CHANG; OU, SHENGQIU; MENG, ZHIBIN; KANG, PING; FAN, LIWEI; QI, FENG; MA, YILONG

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  16. Microarray and Co-expression Network Analysis of Genes Associated with Acute Doxorubicin Cardiomyopathy in Mice.

    PubMed

    Wei, Sheng-Nan; Zhao, Wen-Jie; Zeng, Xiang-Jun; Kang, Yu-Ming; Du, Jie; Li, Hui-Hua

    2015-10-01

    Clinical use of doxorubicin (DOX) in cancer therapy is limited by its dose-dependent cardiotoxicity. But molecular mechanisms underlying this phenomenon have not been well defined. This study was to investigate the effect of DOX on the changes of global genomics in hearts. Acute cardiotoxicity was induced by giving C57BL/6J mice a single intraperitoneal injection of DOX (15 mg/kg). Cardiac function and apoptosis were monitored using echocardiography and TUNEL assay at days 1, 3 and 5. Myocardial glucose and ATP levels were measured. Microarray assays were used to screen gene expression profiles in the hearts at day 5, and the results were confirmed with qPCR analysis. DOX administration caused decreased cardiac function, increased cardiomyocyte apoptosis and decreased glucose and ATP levels. Microarrays showed 747 up-regulated genes and 438 down-regulated genes involved in seven main functional categories. Among them, metabolic pathway was the most affected by DOX. Several key genes, including 2,3-bisphosphoglycerate mutase (Bpgm), hexokinase 2, pyruvate dehydrogenase kinase, isoenzyme 4 and fructose-2,6-bisphosphate 2-phosphatase, are closely related to glucose metabolism. Gene co-expression networks suggested the core role of Bpgm in DOX cardiomyopathy. These results obtained in mice were further confirmed in cultured cardiomyocytes. In conclusion, genes involved in glucose metabolism, especially Bpgm, may play a central role in the pathogenesis of DOX-induced cardiotoxicity. PMID:25575753

  17. Analysis of the dynamic co-expression network of heart regeneration in the zebrafish.

    PubMed

    Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

    2016-01-01

    The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. PMID:27241320

  18. Ligand Similarity Complements Sequence, Physical Interaction, and Co-Expression for Gene Function Prediction

    PubMed Central

    Shoichet, Brian K.; Gillis, Jesse

    2016-01-01

    The expansion of protein-ligand annotation databases has enabled large-scale networking of proteins by ligand similarity. These ligand-based protein networks, which implicitly predict the ability of neighboring proteins to bind related ligands, may complement biologically-oriented gene networks, which are used to predict functional or disease relevance. To quantify the degree to which such ligand-based protein associations might complement functional genomic associations, including sequence similarity, physical protein-protein interactions, co-expression, and disease gene annotations, we calculated a network based on the Similarity Ensemble Approach (SEA: sea.docking.org), where protein neighbors reflect the similarity of their ligands. We also measured the similarity with functional genomic networks over a common set of 1,131 genes, and found that the networks had only small overlaps, which were significant only due to the large scale of the data. Consistent with the view that the networks contain different information, combining them substantially improved Molecular Function prediction within GO (from AUROC~0.63–0.75 for the individual data modalities to AUROC~0.8 in the aggregate). We investigated the boost in guilt-by-association gene function prediction when the networks are combined and describe underlying properties that can be further exploited. PMID:27467773

  19. Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

    PubMed Central

    Ponchon, Luc; Catala, Marjorie; Seijo, Bili; El Khouri, Marguerite; Dardel, Frédéric; Nonin-Lecomte, Sylvie; Tisné, Carine

    2013-01-01

    RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins. PMID:23804766

  20. Understanding developmental and adaptive cues in pine through metabolite profiling and co-expression network analysis

    PubMed Central

    Cañas, Rafael A.; Canales, Javier; Muñoz-Hernández, Carmen; Granados, Jose M.; Ávila, Concepción; García-Martín, María L.; Cánovas, Francisco M.

    2015-01-01

    Conifers include long-lived evergreen trees of great economic and ecological importance, including pines and spruces. During their long lives conifers must respond to seasonal environmental changes, adapt to unpredictable environmental stresses, and co-ordinate their adaptive adjustments with internal developmental programmes. To gain insights into these responses, we examined metabolite and transcriptomic profiles of needles from naturally growing 25-year-old maritime pine (Pinus pinaster L. Aiton) trees over a year. The effect of environmental parameters such as temperature and rain on needle development were studied. Our results show that seasonal changes in the metabolite profiles were mainly affected by the needles’ age and acclimation for winter, but changes in transcript profiles were mainly dependent on climatic factors. The relative abundance of most transcripts correlated well with temperature, particularly for genes involved in photosynthesis or winter acclimation. Gene network analysis revealed relationships between 14 co-expressed gene modules and development and adaptation to environmental stimuli. Novel Myb transcription factors were identified as candidate regulators during needle development. Our systems-based analysis provides integrated data of the seasonal regulation of maritime pine growth, opening new perspectives for understanding the complex regulatory mechanisms underlying conifers’ adaptive responses. Taken together, our results suggest that the environment regulates the transcriptome for fine tuning of the metabolome during development. PMID:25873654

  1. Genetic Network Inference: From Co-Expression Clustering to Reverse Engineering

    NASA Technical Reports Server (NTRS)

    Dhaeseleer, Patrik; Liang, Shoudan; Somogyi, Roland

    2000-01-01

    Advances in molecular biological, analytical, and computational technologies are enabling us to systematically investigate the complex molecular processes underlying biological systems. In particular, using high-throughput gene expression assays, we are able to measure the output of the gene regulatory network. We aim here to review datamining and modeling approaches for conceptualizing and unraveling the functional relationships implicit in these datasets. Clustering of co-expression profiles allows us to infer shared regulatory inputs and functional pathways. We discuss various aspects of clustering, ranging from distance measures to clustering algorithms and multiple-duster memberships. More advanced analysis aims to infer causal connections between genes directly, i.e., who is regulating whom and how. We discuss several approaches to the problem of reverse engineering of genetic networks, from discrete Boolean networks, to continuous linear and non-linear models. We conclude that the combination of predictive modeling with systematic experimental verification will be required to gain a deeper insight into living organisms, therapeutic targeting, and bioengineering.

  2. Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

    PubMed Central

    Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

    2016-01-01

    The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. PMID:27241320

  3. Integrated gene co-expression network analysis in the growth phase of Mycobacterium tuberculosis reveals new potential drug targets.

    PubMed

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Verma, Srikant Prasad; Kumar, Sanjiv; Ramachandran, Srinivasan

    2013-11-01

    We have carried out weighted gene co-expression network analysis of Mycobacterium tuberculosis to gain insights into gene expression architecture during log phase growth. The differentially expressed genes between at least one pair of 11 different M. tuberculosis strains as source of biological variability were used for co-expression network analysis. This data included genes with highest coefficient of variation in expression. Five distinct modules were identified using topological overlap based clustering. All the modules together showed significant enrichment in biological processes: fatty acid biosynthesis, cell membrane, intracellular membrane bound organelle, DNA replication, Quinone biosynthesis, cell shape and peptidoglycan biosynthesis, ribosome and structural constituents of ribosome and transposition. We then extracted the co-expressed connections which were supported either by transcriptional regulatory network or STRING database or high edge weight of topological overlap. The genes trpC, nadC, pitA, Rv3404c, atpA, pknA, Rv0996, purB, Rv2106 and Rv0796 emerged as top hub genes. After overlaying this network on the iNJ661 metabolic network, the reactions catalyzed by 15 highly connected metabolic genes were knocked down in silico and evaluated by Flux Balance Analysis. The results showed that in 12 out of 15 cases, in 11 more than 50% of reactions catalyzed by genes connected through co-expressed connections also had altered fluxes. The modules 'Turquoise', 'Blue' and 'Red' also showed enrichment in essential genes. We could map 152 of the previously known or proposed drug targets in these modules and identified 15 new potential drug targets based on their high degree of co-expressed connections and strong correlation with module eigengenes. PMID:24056838

  4. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons.

    PubMed

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T; Kasperski, Jeffrey; Gierasch, William W; Edvinsson, Lars; Heistad, Donald D; Faraci, Frank M; Russo, Andrew F

    2004-01-30

    We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT(1B/D) agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT(1B/D) receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis. PMID:14715155

  5. Neuronal arithmetic

    PubMed Central

    Silver, R. Angus

    2016-01-01

    The vast computational power of the brain has traditionally been viewed as arising from the complex connectivity of neural networks, in which an individual neuron acts as a simple linear summation and thresholding device. However, recent studies show that individual neurons utilize a wealth of nonlinear mechanisms to transform synaptic input into output firing. These mechanisms can arise from synaptic plasticity, synaptic noise, and somatic and dendritic conductances. This tool kit of nonlinear mechanisms confers considerable computational power on both morphologically simple and more complex neurons, enabling them to perform a range of arithmetic operations on signals encoded in a variety of different ways. PMID:20531421

  6. Evaluation of the Role of G Protein-Coupled Receptor Kinase 3 in Desensitization of Mouse Odorant Receptors in a Mammalian Cell Line and in Olfactory Sensory Neurons

    PubMed Central

    Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi

    2014-01-01

    Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated. PMID:25313015

  7. The Co-Expression Pattern of Odorant Binding Proteins and Olfactory Receptors Identify Distinct Trichoid Sensilla on the Antenna of the Malaria Mosquito Anopheles gambiae

    PubMed Central

    Schultze, Anna; Pregitzer, Pablo; Walter, Marika F.; Woods, Daniel F.; Marinotti, Osvaldo; Breer, Heinz; Krieger, Jürgen

    2013-01-01

    The initial steps of odorant recognition in the insect olfactory system involve odorant binding proteins (OBPs) and odorant receptors (ORs). While large families of OBPs have been identified in the malaria vector A. gambiae, little is known about their expression pattern in the numerous sensory hairs of the female antenna. We applied whole mount fluorescence in Situ hybridization (WM-FISH) and fluorescence immunohistochemistry (WM-FIHC) to investigate the sensilla co-expression of eight A. gambiae OBPs (AgOBPs), most notably AgOBP1 and AgOBP4, which all have abundant transcripts in female antenna. WM-FISH analysis of female antennae using AgOBP-specific probes revealed marked differences in the number of cells expressing each various AgOBPs. Testing combinations of AgOBP probes in two-color WM-FISH resulted in distinct cellular labeling patterns, indicating a combinatorial expression of AgOBPs and revealing distinct AgOBP requirements for various functional sensilla types. WM-FIHC with antisera to AgOBP1 and AgOBP4 confirmed expression of the respective proteins by support cells and demonstrated a location of OBPs within sensilla trichodea. Based on the finding that AgOBP1 and AgOBP4 as well as the receptor type AgOR2 are involved in the recognition of indole, experiments were performed to explore if the AgOBP-types and AgOR2 are co-expressed in distinct olfactory sensilla. Applying two-color WM-FISH with AgOBP-specific probes and probes specific for AgOR2 revealed a close association of support cells bearing transcripts for AgOBP1 and AgOBP4 and neurons with a transcript for the receptor AgOR2. Moreover, combined WM-FISH/-FIHC approaches using an AgOR2-specific riboprobe and AgOBP-specific antisera revealed the expression of the “ligand-matched” AgOBP1, AgOBP4 and AgOR2 to single trichoid hairs. This result substantiates the notion that a specific response to indole is mediated by an interplay of the proteins. PMID:23861970

  8. Locus coeruleus neuronal activity determines proclivity to consume alcohol in a selectively-bred line of rats that readily consumes alcohol.

    PubMed

    West, Charles H K; Boss-Williams, Katherine A; Ritchie, James C; Weiss, Jay M

    2015-11-01

    Sprague-Dawley rats selectively-bred for susceptibility to stress in our laboratory (Susceptible, or SUS rats) voluntarily consume large amounts of alcohol, and amounts that have, as shown here, pharmacological effects, which normal rats will not do. In this paper, we explore neural events in the brain that underlie this propensity to readily consume alcohol. Activity of locus coeruleus neurons (LC), the major noradrenergic cell body concentration in the brain, influences firing of ventral tegmentum dopaminergic cell bodies of the mesocorticolimbic system (VTA-DA neurons), which mediate rewarding aspects of alcohol. We tested the hypothesis that in SUS rats alcohol potently suppresses LC activity to markedly diminish LC-mediated inhibition of VTA-DA neurons, which permits alcohol to greatly increase VTA-DA activity and rewarding aspects of alcohol. Electrophysiological single-unit recording of LC and VTA-DA activity showed that in SUS rats alcohol decreased LC burst firing much more than in normal rats and as a result markedly increased VTA-DA activity in SUS rats while having no such effect in normal rats. Consistent with this, in a behavioral test for reward using conditioned place preference (CPP), SUS rats showed alcohol, given by intraperitoneal (i.p.) injection, to be rewarding. Next, manipulation of LC activity by microinfusion of drugs into the LC region of SUS rats showed that (a) decreasing LC activity increased alcohol intake and increasing LC activity decreased alcohol intake in accord with the formulation described above, and (b) increasing LC activity blocked both the rewarding effect of alcohol in the CPP test and the usual alcohol-induced increase in VTA-DA single-unit activity seen in SUS rats. An important ancillary finding in the CPP test was that an increase in LC activity was rewarding by itself, while a decrease in LC activity was aversive; consequently, effects of LC manipulations on alcohol-related reward in the CPP test were perhaps even

  9. Reprint of: Locus coeruleus neuronal activity determines proclivity to consume alcohol in a selectively-bred line of rats that readily consumes alcohol.

    PubMed

    West, Charles H K; Boss-Williams, Katherine A; Ritchie, James C; Weiss, Jay M

    2016-02-01

    Sprague-Dawley rats selectively-bred for susceptibility to stress in our laboratory (Susceptible, or SUS rats) voluntarily consume large amounts of alcohol, and amounts that have, as shown here, pharmacological effects, which normal rats will not do. In this paper, we explore neural events in the brain that underlie this propensity to readily consume alcohol. Activity of locus coeruleus neurons (LC), the major noradrenergic cell body concentration in the brain, influences firing of ventral tegmentum dopaminergic cell bodies of the mesocorticolimbic system (VTA-DA neurons), which mediate rewarding aspects of alcohol. We tested the hypothesis that in SUS rats alcohol potently suppresses LC activity to markedly diminish LC-mediated inhibition of VTA-DA neurons, which permits alcohol to greatly increase VTA-DA activity and rewarding aspects of alcohol. Electrophysiological single-unit recording of LC and VTA-DA activity showed that in SUS rats alcohol decreased LC burst firing much more than in normal rats and as a result markedly increased VTA-DA activity in SUS rats while having no such effect in normal rats. Consistent with this, in a behavioral test for reward using conditioned place preference (CPP), SUS rats showed alcohol, given by intraperitoneal (i.p.) injection, to be rewarding. Next, manipulation of LC activity by microinfusion of drugs into the LC region of SUS rats showed that (a) decreasing LC activity increased alcohol intake and increasing LC activity decreased alcohol intake in accord with the formulation described above, and (b) increasing LC activity blocked both the rewarding effect of alcohol in the CPP test and the usual alcohol-induced increase in VTA-DA single-unit activity seen in SUS rats. An important ancillary finding in the CPP test was that an increase in LC activity was rewarding by itself, while a decrease in LC activity was aversive; consequently, effects of LC manipulations on alcohol-related reward in the CPP test were perhaps even

  10. High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons

    PubMed Central

    Qiu, Jian; Nestor, Casey C; Zhang, Chunguang; Padilla, Stephanie L; Palmiter, Richard D

    2016-01-01

    Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1ARH) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1ARH neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1ARH neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1ARH neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1ARH neurons. We propose that Kiss1ARH neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function. DOI: http://dx.doi.org/10.7554/eLife.16246.001 PMID:27549338

  11. High-frequency stimulation-induced peptide release synchronizes arcuate kisspeptin neurons and excites GnRH neurons.

    PubMed

    Qiu, Jian; Nestor, Casey C; Zhang, Chunguang; Padilla, Stephanie L; Palmiter, Richard D; Kelly, Martin J; Rønnekleiv, Oline K

    2016-01-01

    Kisspeptin (Kiss1) and neurokinin B (NKB) neurocircuits are essential for pubertal development and fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (Kiss1(ARH)) co-express Kiss1, NKB, dynorphin and glutamate and are postulated to provide an episodic, excitatory drive to gonadotropin-releasing hormone 1 (GnRH) neurons, the synaptic mechanisms of which are unknown. We characterized the cellular basis for synchronized Kiss1(ARH) neuronal activity using optogenetics, whole-cell electrophysiology, molecular pharmacology and single cell RT-PCR in mice. High-frequency photostimulation of Kiss1(ARH) neurons evoked local release of excitatory (NKB) and inhibitory (dynorphin) neuropeptides, which were found to synchronize the Kiss1(ARH) neuronal firing. The light-evoked synchronous activity caused robust excitation of GnRH neurons by a synaptic mechanism that also involved glutamatergic input to preoptic Kiss1 neurons from Kiss1(ARH) neurons. We propose that Kiss1(ARH) neurons play a dual role of driving episodic secretion of GnRH through the differential release of peptide and amino acid neurotransmitters to coordinate reproductive function. PMID:27549338

  12. Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

    PubMed Central

    Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

    2009-01-01

    Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

  13. Co-expression of three opsins in cone photoreceptors of the salamander, Ambystoma tigrinum

    PubMed Central

    Isayama, Tomoki; Chen, Ying; Kono, Masahiro; Fabre, Eduard; Slavsky, Michael; DeGrip, Willem J.; Ma, Jian-Xing; Crouch, Rosalie K.; Makino, Clint L.

    2014-01-01

    Whereas more than one type of visual opsin is present in the retina of most vertebrates, it was thought that each type of photoreceptor expressed only one opsin. However, evidence has accumulated that some photoreceptors contain more than one opsin, in many cases as a result of a developmental transition from the expression of one opsin to another. The salamander UV-sensitive (UV) cone is particularly notable because it contains three opsins (Makino and Dodd, 1996; J Gen Physiol 108:27–34). Two opsin types are expressed at levels more than a hundred times lower than that of the primary opsin. Here, immunohistochemical experiments identified the primary component as a UV cone opsin and the two minor components as the short wavelength-sensitive (S) and long wavelength-sensitive (L) cone opsins. Based on single cell recordings of 156 photoreceptors, the presence of three components in UV cones of hatchlings and terrestrial adults ruled out a developmental transition. There was no evidence for multiple opsin types within rods or S cones. But immunohistochemistry and partial bleaching in conjunction with single cell recording revealed that both single and double L cones contained low levels of short wavelength-sensitive pigments in addition to the main L visual pigment. These results raise the possibility that co-expression of multiple opsins in other vertebrates was overlooked because a minor component absorbing at short wavelengths was masked by the main visual pigment or because the expression level of a component absorbing at long wavelengths was exceedingly low. PMID:24374736

  14. 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

  15. Increased fiber outgrowth from xeno-transplanted human embryonic dopaminergic neurons with co-implants of polymer-encapsulated genetically modified cells releasing glial cell line-derived neurotrophic factor.

    PubMed

    Ahn, Young-Hwan; Bensadoun, Jean-Charles; Aebischer, Patrick; Zurn, Anne D; Seiger, Ake; Björklund, Anders; Lindvall, Olle; Wahlberg, Lars; Brundin, Patrik; Kaminski Schierle, Gabriele S

    2005-07-30

    We investigated whether a continuous supply of glial cell line-derived neurotrophic factor (GDNF) via encapsulated genetically modified cells can promote survival and fiber outgrowth from xenotransplanted human dopaminergic neurons. Cells genetically engineered to continuously secrete GDNF were confined in hollow fiber-based macrocapsules. Each hemiparkinsonian rat received either a single C2C12-hGDNF capsule (n=8) or a C2C12-control capsule (n=8) concomitantly with human embryonic ventral mesencephalic cell suspension transplants. Our results show that fiber outgrowth in the area between the capsule and the graft is more extensive in rats with GDNF-releasing capsules than in rats with control capsules. We suggest that continuous and safe delivery of GDNF to the brain could be a potential way to optimize neural transplantation as a therapy for Parkinson's disease. PMID:15982530

  16. Analysis of two P-element enhancer-trap insertion lines that show expression in the giant fibre neuron of Drosophila melanogaster.

    PubMed

    Allen, M J; Drummond, J A; Sweetman, D J; Moffat, K G

    2007-06-01

    The giant fibre system (GFS) of Drosophila is a simple neural circuit that mediates escape responses in adult flies. Here we report the initial characterization of two genes that are preferentially expressed in the GFS. Two P-element insertion lines, carrying the GAL4 transcriptional activator, were identified that exhibited pronounced expression in elements of the GFS and relatively low levels elsewhere within the adult central nervous system. Genomic DNA flanking the P-element insertion site was recovered from each of these lines, sequenced, and nearby transcripts identified and confirmed to exhibit GFS expression by in situ hybridization. This analysis revealed that these P-elements were in previously characterized genes. Line P[GAL4]-A307 has an insert in the gene short stop for which we have identified a novel transcript, while line P[GAL4]-141 has an insert in the transcription factor ken and barbie. Here we show that ken and barbie mutants have defects in escape behaviour, behavioural responses to visual stimuli and synaptic functions in the GFS. We have therefore revealed a neural role for a transcription factor that previously had no implicated neural function. PMID:16879616

  17. Recursive Indirect-Paths Modularity (RIP-M) for Detecting Community Structure in RNA-Seq Co-expression Networks.

    PubMed

    Rahmani, Bahareh; Zimmermann, Michael T; Grill, Diane E; Kennedy, Richard B; Oberg, Ann L; White, Bill C; Poland, Gregory A; McKinney, Brett A

    2016-01-01

    Clusters of genes in co-expression networks are commonly used as functional units for gene set enrichment detection and increasingly as features (attribute construction) for statistical inference and sample classification. One of the practical challenges of clustering for these purposes is to identify an optimal partition of the network where the individual clusters are neither too large, prohibiting interpretation, nor too small, precluding general inference. Newman Modularity is a spectral clustering algorithm that automatically finds the number of clusters, but for many biological networks the cluster sizes are suboptimal. In this work, we generalize Newman Modularity to incorporate information from indirect paths in RNA-Seq co-expression networks. We implement a merge-and-split algorithm that allows the user to constrain the range of cluster sizes: large enough to capture genes in relevant pathways, yet small enough to resolve distinct functions. We investigate the properties of our recursive indirect-pathways modularity (RIP-M) and compare it with other clustering methods using simulated co-expression networks and RNA-seq data from an influenza vaccine response study. RIP-M had higher cluster assignment accuracy than Newman Modularity for finding clusters in simulated co-expression networks for all scenarios, and RIP-M had comparable accuracy to Weighted Gene Correlation Network Analysis (WGCNA). RIP-M was more accurate than WGCNA for modest hard thresholds and comparable for high, while WGCNA was slightly more accurate for soft thresholds. In the vaccine study data, RIP-M and WGCNA enriched for a comparable number of immunologically relevant pathways. PMID:27242890

  18. Recursive Indirect-Paths Modularity (RIP-M) for Detecting Community Structure in RNA-Seq Co-expression Networks

    PubMed Central

    Rahmani, Bahareh; Zimmermann, Michael T.; Grill, Diane E.; Kennedy, Richard B.; Oberg, Ann L.; White, Bill C.; Poland, Gregory A.; McKinney, Brett A.

    2016-01-01

    Clusters of genes in co-expression networks are commonly used as functional units for gene set enrichment detection and increasingly as features (attribute construction) for statistical inference and sample classification. One of the practical challenges of clustering for these purposes is to identify an optimal partition of the network where the individual clusters are neither too large, prohibiting interpretation, nor too small, precluding general inference. Newman Modularity is a spectral clustering algorithm that automatically finds the number of clusters, but for many biological networks the cluster sizes are suboptimal. In this work, we generalize Newman Modularity to incorporate information from indirect paths in RNA-Seq co-expression networks. We implement a merge-and-split algorithm that allows the user to constrain the range of cluster sizes: large enough to capture genes in relevant pathways, yet small enough to resolve distinct functions. We investigate the properties of our recursive indirect-pathways modularity (RIP-M) and compare it with other clustering methods using simulated co-expression networks and RNA-seq data from an influenza vaccine response study. RIP-M had higher cluster assignment accuracy than Newman Modularity for finding clusters in simulated co-expression networks for all scenarios, and RIP-M had comparable accuracy to Weighted Gene Correlation Network Analysis (WGCNA). RIP-M was more accurate than WGCNA for modest hard thresholds and comparable for high, while WGCNA was slightly more accurate for soft thresholds. In the vaccine study data, RIP-M and WGCNA enriched for a comparable number of immunologically relevant pathways. PMID:27242890

  19. Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression.

    PubMed

    Tong, Yanjun; Feng, Shoushuai; Xin, Yu; Yang, Hailin; Zhang, Ling; Wang, Wu; Chen, Wei

    2016-01-20

    The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli. The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43 kDa protein band, indicating SOX was successfully expressed in E. coli. However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in "inclusion bodies" owing to the "hot spots" of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli. PMID:26626227

  20. A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

    PubMed

    Chanda, Pranab K; Edris, Wade A; Kennedy, Jeffrey D

    2006-05-01

    A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies. PMID:16325426

  1. A co-expression network analysis reveals lncRNA abnormalities in peripheral blood in early-onset schizophrenia.

    PubMed

    Ren, Yan; Cui, Yuehua; Li, Xinrong; Wang, Binhong; Na, Long; Shi, Junyan; Wang, Liang; Qiu, Lixia; Zhang, Kerang; Liu, Guifen; Xu, Yong

    2015-12-01

    Long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression and disease processes especially in neuropsychiatric disorders. To explore the potential regulatory roles of lncRNAs in schizophrenia, we performed an integrated co-expression network analysis on lncRNA and mRNA microarray profiles generated from the peripheral blood samples in 19 drug-naïve first-episode early-onset schizophrenia (EOS) patients and 18 demographically matched typically developing controls (TDCs). Using weighted gene co-expression network analysis (WGCNA), we showed that the lncRNAs were organized into co-expressed modules, and two lncRNA modules were associated with EOS. The mRNA networks were constructed and three disease-associated modules were identified. Gene Ontology (GO) analysis indicated that the mRNAs were highly enriched for mitochondrion and related biological processes. Moreover, our results revealed a significant correlation between lncRNAs and mRNAs using the canonical correlation analysis (CCA). Our results suggest that the convergent lncRNA alteration may be involved in the etiologies of EOS, and mitochondrial dysfunction participates in the pathological process of the disease. Our findings may shed light on the pathogenesis of schizophrenia and facilitate future diagnosis and therapeutic strategies. PMID:25967042

  2. Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.

    PubMed

    Tang, Gong-Li; Yang, Chun-Song; Bao, Jian-Shao; Wang, Yan-Fang; Chen, Hai-Bao; Shi, Ding-Ji; Liu, Feng-Long

    2001-01-01

    To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained. PMID:12053203

  3. [Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A].

    PubMed

    Guo, Jian-Qiang; Yao, Li-Hong; Chen, Ai-Jun; Xu, Yi; Jia, Run-Qing; Bo, Hong; Dong, Jie; Zhou, Jian-Fang; Shu, Yue-Long; Zhang, Zhi-Qing

    2009-03-01

    Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector. PMID:19678564

  4. Leptin modulates nutrient reward via inhibitory galanin action on orexin neurons

    PubMed Central

    Laque, Amanda; Yu, Sangho; Qualls-Creekmore, Emily; Gettys, Sarah; Schwartzenburg, Candice; Bui, Kelly; Rhodes, Christopher; Berthoud, Hans-Rudolf; Morrison, Christopher D.; Richards, Brenda K.; Münzberg, Heike

    2015-01-01

    Objective Leptin modulates food reward via central leptin receptor (LepRb) expressing neurons. Food reward requires stimulation of midbrain dopamine neurons and is modulated by central leptin action, but the exact central mechanisms remain unclear. Stimulatory and inhibitory leptin actions on dopamine neurons have been reported, e.g. by indirect actions on orexin neurons or via direct innervation of dopamine neurons in the ventral tegmental area. Methods We showed earlier that LepRb neurons in the lateral hypothalamus (LHA) co-express the inhibitory acting neuropeptide galanin (GAL-LepRb neurons). We studied the involvement of GAL-LepRb neurons to regulate nutrient reward in mice with selective LepRb deletion from galanin neurons (GAL-LepRbKO mice). Results We found that the rewarding value and preference for sucrose over fat was increased in GAL-LepRbKO mice compared to controls. LHA GAL-LepRb neurons innervate orexin neurons, but not the VTA. Further, expression of galanin and its receptor GalR1 are decreased in the LHA of GAL-LepRbKO mice, resulting in increased activation of orexin neurons. Conclusion We suggest galanin as an important mediator of leptin action to modulate nutrient reward by inhibiting orexin neurons. PMID:26500842

  5. Optical control of neuronal excitation and inhibition using a single opsin protein, ChR2

    PubMed Central

    Liske, Holly; Qian, Xiang; Anikeeva, Polina; Deisseroth, Karl; Delp, Scott

    2013-01-01

    The effect of electrical stimulation on neuronal membrane potential is frequency dependent. Low frequency electrical stimulation can evoke action potentials, whereas high frequency stimulation can inhibit action potential transmission. Optical stimulation of channelrhodopsin-2 (ChR2) expressed in neuronal membranes can also excite action potentials. However, it is unknown whether optical stimulation of ChR2-expressing neurons produces a transition from excitation to inhibition with increasing light pulse frequencies. Here we report optical inhibition of motor neuron and muscle activity in vivo in the cooled sciatic nerves of Thy1-ChR2-EYFP mice. We also demonstrate all-optical single-wavelength control of neuronal excitation and inhibition without co-expression of inhibitory and excitatory opsins. This all-optical system is free from stimulation-induced electrical artifacts and thus provides a new approach to investigate mechanisms of high frequency inhibition in neuronal circuits in vivo and in vitro. PMID:24173561

  6. L-citrulline immunostaining identifies nitric oxide production sites within neurons

    NASA Technical Reports Server (NTRS)

    Martinelli, G. P. T.; Friedrich, V. L. Jr; Holstein, G. R.

    2002-01-01

    The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker. Copyright 2002 IBRO.

  7. Motor Neuron Diseases

    MedlinePlus

    ... Enhancing Diversity Find People About NINDS NINDS Motor Neuron Diseases Information Page Condensed from Motor Neuron Diseases ... and Information Publicaciones en Español What are Motor Neuron Diseases? The motor neuron diseases (MNDs) are a ...

  8. Motor Neuron Diseases

    MedlinePlus

    ... called upper motor neurons ) are transmitted to nerve cells in the brain stem and spinal cord (called lower motor neurons ) and from them to particular muscles. Upper motor neurons direct the lower motor neurons ...

  9. Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

    PubMed

    Kim, Ju-Kon; Jang, In-Cheol; Wu, Ray; Zuo, Wei-Neng; Boston, Rebecca S; Lee, Yong-Hwan; Ahn, Il-Pyung; Nahm, Baek Hie

    2003-08-01

    Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression. PMID:12885168

  10. Construction of a compatible Gateway-based co-expression vector set for expressing multiprotein complexes in E. coli.

    PubMed

    Salim, Loubna; Feger, Claire; Busso, Didier

    2016-11-01

    We report the construction of a versatile Gateway-based co-expression vector set for producing multiprotein complexes in Escherichia coli. The set consists of two groups of three vectors (pCoGW and pCo0GW), each having a specific antibiotic resistance gene, a compatible origin of replication and allowing cloning of up to two genes, each under control of its own T7 promoter. To validate the set, 33 (co-)expression plasmids encoding fluorescent protein (GFP, DsRed and ECFP) have been generated. Protein expression levels were quantified and (co-)expression visualized by fluorescent microscopy. The results illustrate the applicability of these vectors in co-expression studies. PMID:27558914

  11. Deciphering a neuronal circuit that mediates loss of appetite

    PubMed Central

    Wu, Qi; Clark, Michael S.; Palmiter, Richard D.

    2014-01-01

    Hypothalamic neurons that co-express agouti-related protein (AgRP), neuropeptide Y (NPY), and γ-amino-butyric acid (GABA) are known to promote feeding and weight gain by integration of various nutritional, hormonal, and neuronal signals1,2. Ablation of these neurons leads to cessation of feeding that is accompanied by Fos activation in most regions where they project3–6. Previous experiments indicate that the ensuing starvation is due to aberrant activation of the parabrachial nucleus (PBN) and it could be prevented by facilitating GABAA receptor signaling in the PBN within a critical adaptation period5. We hypothesized that loss of GABAergic inhibition from AgRP neurons to the PBN leads to abnormal activation of the PBN, which in turn inhibits feeding. However, the source of the excitatory inputs to the PBN was unknown. Here we show that glutamatergic neurons in the nucleus tractus solitarius (NTS) and caudal serotonergic neurons control the excitability of PBN neurons and inhibit feeding. Blockade of 5-HT3 receptor signaling in the rostral NTS by either chronic administration of ondansetron or genetic inactivation of Tph2 in caudal serotonergic neurons that project to the NTS protects against starvation when AgRP neurons are ablated. Moreover, genetic inactivation of glutamatergic signaling by the NTS onto N-methyl D-aspartate (NMDA)-type glutamate receptors in the PBN prevents starvation. We also demonstrate that suppressing glutamatergic output of the PBN reinstates normal appetite after AgRP neuron ablation, whereas it promotes weight gain without AgRP neuron ablation. Hence, we identify the PBN as an important hub that integrates signals from several brain regions to bidirectionally modulate feeding and body weight. PMID:22419158

  12. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    PubMed Central

    2010-01-01

    Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is

  13. Over-producing soluble protein complex and validating protein-protein interaction through a new bacterial co-expression system.

    PubMed

    Zeng, Jumei; Zhang, Lei; Li, Yuqing; Wang, Yi; Wang, Mingchao; Duan, Xin; He, Zheng-Guo

    2010-01-01

    Many proteins exert their functions through a protein complex and protein-protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein-protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein-protein interaction of interest in a wide range of biological organisms. PMID:19747546

  14. Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

    PubMed Central

    2012-01-01

    Background Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions. Results Recombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%. Conclusion Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as

  15. Gene co-expression network analysis in Rhodobacter capsulatus and application to comparative expression analysis of Rhodobacter sphaeroides

    SciTech Connect

    Pena-Castillo, Lourdes; Mercer, Ryan; Gurinovich, Anastasia; Callister, Stephen J.; Wright, Aaron T.; Westbye, Alexander; Beatty, J. T.; Lang, Andrew S.

    2014-08-28

    The genus Rhodobacter contains purple nonsulfur bacteria found mostly in freshwater environments. Representative strains of two Rhodobacter species, R. capsulatus and R. sphaeroides, have had their genomes fully sequenced and both have been the subject of transcriptional profiling studies. Gene co-expression networks can be used to identify modules of genes with similar expression profiles. Functional analysis of gene modules can then associate co-expressed genes with biological pathways, and network statistics can determine the degree of module preservation in related networks. In this paper, we constructed an R. capsulatus gene co-expression network, performed functional analysis of identified gene modules, and investigated preservation of these modules in R. capsulatus proteomics data and in R. sphaeroides transcriptomics data. Results: The analysis identified 40 gene co-expression modules in R. capsulatus. Investigation of the module gene contents and expression profiles revealed patterns that were validated based on previous studies supporting the biological relevance of these modules. We identified two R. capsulatus gene modules preserved in the protein abundance data. We also identified several gene modules preserved between both Rhodobacter species, which indicate that these cellular processes are conserved between the species and are candidates for functional information transfer between species. Many gene modules were non-preserved, providing insight into processes that differentiate the two species. In addition, using Local Network Similarity (LNS), a recently proposed metric for expression divergence, we assessed the expression conservation of between-species pairs of orthologs, and within-species gene-protein expression profiles. Conclusions: Our analyses provide new sources of information for functional annotation in R. capsulatus because uncharacterized genes in modules are now connected with groups of genes that constitute a joint functional

  16. Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking

    SciTech Connect

    Shankaran, Harish; Zhang, Yi; Opresko, Lee; Resat, Haluk

    2008-06-27

    The integration of experimental measurements and computational predictions is a powerful means to understand the molecular mechanisms of complex biological systems. The human epidermal growth factor receptor (HER) system is an intricately regulated system that plays critical roles in development and tumorigenesis. Here, we apply integrated experimentation and modeling to analyze HER receptor activation in a panel of cell lines expressing different levels of HER1 and HER2. A mathematical model that includes the fundamental biochemical/biophysical processes involved in receptor activation was used to fit the experimental data, and 19 independent parameters including receptor dimerization affinities, trafficking rates and relative phosphorylation levels were estimated. The parameter values quantitatively support existing ideas on the effect of HER2 on HER1 phosphorylation dynamics, and enable us to predict receptor dimerization patterns in the cell lines. The integrated approach described here can be applied to obtain a predictive understanding of other biomolecular systems.

  17. Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity.

    PubMed

    Gu, Yang-Zhuo; Fan, Chuan-Wen; Lu, Ran; Shao, Bin; Sang, Ya-Xiong; Huang, Qiao-Rong; Li, Xue; Meng, Wen-Tong; Mo, Xian-Ming; Wei, Yu-Quan

    2016-01-01

    Genetic modification of whole-cell cancer vaccines to augment their efficacies has a history of over two and a half decades. Various genes and gene combinations, targeting different aspects of immune responses have been tested in pursuit of potent adjuvant effects. Here we show that co-expression of two cytokine members of the common cytokine receptor γ-chain family, IL-21 and IL-7, in whole-cell cancer vaccines boosts antitumor immunity in a CD4(+) and CD8(+) T cell-dependent fashion. It also generates effective immune memory. The vaccine-elicited short-term effects positively correlated with enhanced infiltration of CD4(+) and CD8(+) effector T cells, and the long-term effects positively correlated with enhanced infiltration of effector memory T cells, especially CD8(+) effector memory T cells. Preliminary data suggested that the vaccine exhibited good safety profile in murine models. Taken together, the combination of IL-21 and IL-7 possesses potent adjuvant efficacy in whole-cell vaccines. This finding warrants future development of IL-21 and IL-7 co-expressing whole-cell cancer vaccines and their relevant combinatorial regimens. PMID:27571893

  18. [Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1].

    PubMed

    Chen, Fei; Hu, Meirong; Jiang, Xianzhang; Tao, Yong; Huang, Jianzhong

    2015-12-01

    The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL. PMID:27093831

  19. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

    PubMed

    Wang, Rongzhi; Xiang, Shuangshuang; Feng, Youjun; Srinivas, Swaminath; Zhang, Yonghui; Lin, Mingshen; Wang, Shihua

    2013-01-01

    Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody. PMID:24224158

  20. Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

    PubMed

    Prokhnevsky, Alex; Mamedov, Tarlan; Leffet, Brett; Rahimova, Rahila; Ghosh, Ananya; Mett, Vadim; Yusibov, Vidadi

    2015-02-01

    In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell. PMID:25280556

  1. Identification of candidate genes in Populus cell wall biosynthesis using text-mining, co-expression network and comparative genomics

    SciTech Connect

    Yang, Xiaohan; Ye, Chuyu; Bisaria, Anjali; Tuskan, Gerald A; Kalluri, Udaya C

    2011-01-01

    Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of ethanol from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidences supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional genomics in relation to cell wall biosynthesis.

  2. Paramyxovirus mediated cell fusion requires co-expression of both the fusion and hemagglutinin-neuraminidase glycoproteins.

    PubMed

    Heminway, B R; Yu, Y; Galinski, M S

    1994-01-01

    Syncytia formation in either CV-1 or HeLa T4+ cells required recombinant expression of both fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the human parainfluenza virus type 3 (HPIV3), human parainfluenza virus type 2 (HPIV2), and simian virus 5 (SV5). In this system, recombinant T7 transcription vectors (pT7-5 or pGEM) containing F or HN, were transfected individually or in combination into cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3). While both proteins were processed and expressed at the cell surface, syncytia formation occurred only when both glycoproteins were co-expressed. The function of HN in the fusion process could not be replaced using lectins or by co-expression of heterologous F and HN proteins. Further, cell fusion was not observed when experiments were performed using individually expressed F and HN proteins in adjacent cells. The data presented in this report support the notion that a specific interaction between both paramyxoviral glycoproteins is required for the formation of syncytia in tissue culture monolayers. PMID:8165862

  3. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  4. Quantitative proteomics reveals the novel co-expression signatures in early brain development for prognosis of glioblastoma multiforme

    PubMed Central

    Yu, Xuexin; Feng, Lin; Liu, Dianming; Zhang, Lianfeng; Wu, Bo; Jiang, Wei; Han, Zujing; Cheng, Shujun

    2016-01-01

    Although several researches have explored the similarity across development and tumorigenesis in cellular behavior and underlying molecular mechanisms, not many have investigated the developmental characteristics at proteomic level and further extended to cancer clinical outcome. In this study, we used iTRAQ to quantify the protein expression changes during macaque rhesus brain development from fetuses at gestation 70 days to after born 5 years. Then, we performed weighted gene co-expression network analysis (WGCNA) on protein expression data of brain development to identify co-expressed modules that highly expressed on distinct development stages, including early stage, middle stage and late stage. Moreover, we used the univariate cox regression model to evaluate the prognostic potentials of these genes in two independent glioblastoma multiforme (GBM) datasets. The results showed that the modules highly expressed on early stage contained more reproducible prognostic genes, including ILF2, CCT7, CCT4, RPL10A, MSN, PRPS1, TFRC and APEX1. These genes were not only associated with clinical outcome, but also tended to influence chemoresponse. These signatures identified from embryonic brain development might contribute to precise prediction of GBM prognosis and identification of novel drug targets in GBM therapies. Thus, the development could become a viable reference model for researching cancers, including identifying novel prognostic markers and promoting new therapies. PMID:26895104

  5. Gene Co-Expression Network Analysis for Identifying Modules and Functionally Enriched Pathways in Type 1 Diabetes.

    PubMed

    Riquelme Medina, Ignacio; Lubovac-Pilav, Zelmina

    2016-01-01

    Type 1 diabetes (T1D) is a complex disease, caused by the autoimmune destruction of the insulin producing pancreatic beta cells, resulting in the body's inability to produce insulin. While great efforts have been put into understanding the genetic and environmental factors that contribute to the etiology of the disease, the exact molecular mechanisms are still largely unknown. T1D is a heterogeneous disease, and previous research in this field is mainly focused on the analysis of single genes, or using traditional gene expression profiling, which generally does not reveal the functional context of a gene associated with a complex disorder. However, network-based analysis does take into account the interactions between the diabetes specific genes or proteins and contributes to new knowledge about disease modules, which in turn can be used for identification of potential new biomarkers for T1D. In this study, we analyzed public microarray data of T1D patients and healthy controls by applying a systems biology approach that combines network-based Weighted Gene Co-Expression Network Analysis (WGCNA) with functional enrichment analysis. Novel co-expression gene network modules associated with T1D were elucidated, which in turn provided a basis for the identification of potential pathways and biomarker genes that may be involved in development of T1D. PMID:27257970

  6. Quantitative proteomics reveals the novel co-expression signatures in early brain development for prognosis of glioblastoma multiforme.

    PubMed

    Yu, Xuexin; Feng, Lin; Liu, Dianming; Zhang, Lianfeng; Wu, Bo; Jiang, Wei; Han, Zujing; Cheng, Shujun

    2016-03-22

    Although several researches have explored the similarity across development and tumorigenesis in cellular behavior and underlying molecular mechanisms, not many have investigated the developmental characteristics at proteomic level and further extended to cancer clinical outcome. In this study, we used iTRAQ to quantify the protein expression changes during macaque rhesus brain development from fetuses at gestation 70 days to after born 5 years. Then, we performed weighted gene co-expression network analysis (WGCNA) on protein expression data of brain development to identify co-expressed modules that highly expressed on distinct development stages, including early stage, middle stage and late stage. Moreover, we used the univariate cox regression model to evaluate the prognostic potentials of these genes in two independent glioblastoma multiforme (GBM) datasets. The results showed that the modules highly expressed on early stage contained more reproducible prognostic genes, including ILF2, CCT7, CCT4, RPL10A, MSN, PRPS1, TFRC and APEX1. These genes were not only associated with clinical outcome, but also tended to influence chemoresponse. These signatures identified from embryonic brain development might contribute to precise prediction of GBM prognosis and identification of novel drug targets in GBM therapies. Thus, the development could become a viable reference model for researching cancers, including identifying novel prognostic markers and promoting new therapies. PMID:26895104

  7. Integration of Metabolic Modeling with Gene Co-expression Reveals Transcriptionally Programmed Reactions Explaining Robustness in Mycobacterium tuberculosis

    PubMed Central

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Mittal, Inna; Mobeen, Ahmed; Ramachandran, Srinivasan

    2016-01-01

    Robustness of metabolic networks is accomplished by gene regulation, modularity, re-routing of metabolites and plasticity. Here, we probed robustness against perturbations of biochemical reactions of M. tuberculosis in the form of predicting compensatory trends. In order to investigate the transcriptional programming of genes associated with correlated fluxes, we integrated with gene co-expression network. Knock down of the reactions NADH2r and ATPS responsible for producing the hub metabolites, and Central carbon metabolism had the highest proportion of their associated genes under transcriptional co-expression with genes of their flux correlated reactions. Reciprocal gene expression correlations were observed among compensatory routes, fresh activation of alternative routes and in the multi-copy genes of Cysteine synthase and of Phosphate transporter. Knock down of 46 reactions caused the activation of Isocitrate lyase or Malate synthase or both reactions, which are central to the persistent state of M. tuberculosis. A total of 30 new freshly activated routes including Cytochrome c oxidase, Lactate dehydrogenase, and Glycine cleavage system were predicted, which could be responsible for switching into dormant or persistent state. Thus, our integrated approach of exploring transcriptional programming of flux correlated reactions has the potential to unravel features of system architecture conferring robustness. PMID:27000948

  8. MCT1 modulates cancer cell pyruvate export and growth of tumors that co-express MCT1 and MCT4

    PubMed Central

    Hong, Candice Sun; Graham, Nicholas A.; Gu, Wen; Camacho, Carolina Espindola; Mah, Vei; Maresh, Erin L.; Alavi, Mohammed; Bagryanova, Lora; Krotee, Pascal A. L.; Gardner, Brian K.; Behbahan, Iman Saramipoor; Horvath, Steve; Chia, David; Mellinghoff, Ingo K.; Hurvitz, Sara A.; Dubinett, Steven M.; Critchlow, Susan E.; Kurdistani, Siavash K.; Goodglick, Lee; Braas, Daniel; Graeber, Thomas G.; Christofk, Heather R.

    2016-01-01

    SUMMARY Monocarboxylate Transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export, which when inhibited enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors further supporting their use as anti-cancer therapeutics. PMID:26876179

  9. Gene Co-Expression Network Analysis for Identifying Modules and Functionally Enriched Pathways in Type 1 Diabetes

    PubMed Central

    Riquelme Medina, Ignacio; Lubovac-Pilav, Zelmina

    2016-01-01

    Type 1 diabetes (T1D) is a complex disease, caused by the autoimmune destruction of the insulin producing pancreatic beta cells, resulting in the body’s inability to produce insulin. While great efforts have been put into understanding the genetic and environmental factors that contribute to the etiology of the disease, the exact molecular mechanisms are still largely unknown. T1D is a heterogeneous disease, and previous research in this field is mainly focused on the analysis of single genes, or using traditional gene expression profiling, which generally does not reveal the functional context of a gene associated with a complex disorder. However, network-based analysis does take into account the interactions between the diabetes specific genes or proteins and contributes to new knowledge about disease modules, which in turn can be used for identification of potential new biomarkers for T1D. In this study, we analyzed public microarray data of T1D patients and healthy controls by applying a systems biology approach that combines network-based Weighted Gene Co-Expression Network Analysis (WGCNA) with functional enrichment analysis. Novel co-expression gene network modules associated with T1D were elucidated, which in turn provided a basis for the identification of potential pathways and biomarker genes that may be involved in development of T1D. PMID:27257970

  10. Integration of Metabolic Modeling with Gene Co-expression Reveals Transcriptionally Programmed Reactions Explaining Robustness in Mycobacterium tuberculosis.

    PubMed

    Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Mittal, Inna; Mobeen, Ahmed; Ramachandran, Srinivasan

    2016-01-01

    Robustness of metabolic networks is accomplished by gene regulation, modularity, re-routing of metabolites and plasticity. Here, we probed robustness against perturbations of biochemical reactions of M. tuberculosis in the form of predicting compensatory trends. In order to investigate the transcriptional programming of genes associated with correlated fluxes, we integrated with gene co-expression network. Knock down of the reactions NADH2r and ATPS responsible for producing the hub metabolites, and Central carbon metabolism had the highest proportion of their associated genes under transcriptional co-expression with genes of their flux correlated reactions. Reciprocal gene expression correlations were observed among compensatory routes, fresh activation of alternative routes and in the multi-copy genes of Cysteine synthase and of Phosphate transporter. Knock down of 46 reactions caused the activation of Isocitrate lyase or Malate synthase or both reactions, which are central to the persistent state of M. tuberculosis. A total of 30 new freshly activated routes including Cytochrome c oxidase, Lactate dehydrogenase, and Glycine cleavage system were predicted, which could be responsible for switching into dormant or persistent state. Thus, our integrated approach of exploring transcriptional programming of flux correlated reactions has the potential to unravel features of system architecture conferring robustness. PMID:27000948

  11. Increased co-expression of genes harboring the damaging de novo mutations in Chinese schizophrenic patients during prenatal development

    PubMed Central

    Wang, Qiang; Li, Miaoxin; Yang, Zhenxing; Hu, Xun; Wu, Hei-Man; Ni, Peiyan; Ren, Hongyan; Deng, Wei; Li, Mingli; Ma, Xiaohong; Guo, Wanjun; Zhao, Liansheng; Wang, Yingcheng; Xiang, Bo; Lei, Wei; Sham, Pak C; Li, Tao

    2015-01-01

    Schizophrenia is a heritable, heterogeneous common psychiatric disorder. In this study, we evaluated the hypothesis that de novo variants (DNVs) contribute to the pathogenesis of schizophrenia. We performed exome sequencing in Chinese patients (N = 45) with schizophrenia and their unaffected parents (N = 90). Forty genes were found to contain DNVs. These genes had enriched transcriptional co-expression profile in prenatal frontal cortex (Bonferroni corrected p < 9.1 × 10−3), and in prenatal temporal and parietal regions (Bonferroni corrected p < 0.03). Also, four prenatal anatomical subregions (VCF, MFC, OFC and ITC) have shown significant enrichment of connectedness in co-expression networks. Moreover, four genes (LRP1, MACF1, DICER1 and ABCA2) harboring the damaging de novo mutations are strongly prioritized as susceptibility genes by multiple evidences. Our findings in Chinese schizophrenic patients indicate the pathogenic role of DNVs, supporting the hypothesis that schizophrenia is a neurodevelopmental disease. PMID:26666178

  12. Forced co-expression of IL-21 and IL-7 in whole-cell cancer vaccines promotes antitumor immunity

    PubMed Central

    Gu, Yang-Zhuo; Fan, Chuan-Wen; Lu, Ran; Shao, Bin; Sang, Ya-Xiong; Huang, Qiao-Rong; Li, Xue; Meng, Wen-Tong; Mo, Xian-Ming; Wei, Yu-Quan

    2016-01-01

    Genetic modification of whole-cell cancer vaccines to augment their efficacies has a history of over two and a half decades. Various genes and gene combinations, targeting different aspects of immune responses have been tested in pursuit of potent adjuvant effects. Here we show that co-expression of two cytokine members of the common cytokine receptor γ-chain family, IL-21 and IL-7, in whole-cell cancer vaccines boosts antitumor immunity in a CD4+ and CD8+ T cell-dependent fashion. It also generates effective immune memory. The vaccine-elicited short-term effects positively correlated with enhanced infiltration of CD4+ and CD8+ effector T cells, and the long-term effects positively correlated with enhanced infiltration of effector memory T cells, especially CD8+ effector memory T cells. Preliminary data suggested that the vaccine exhibited good safety profile in murine models. Taken together, the combination of IL-21 and IL-7 possesses potent adjuvant efficacy in whole-cell vaccines. This finding warrants future development of IL-21 and IL-7 co-expressing whole-cell cancer vaccines and their relevant combinatorial regimens. PMID:27571893

  13. Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders.

    PubMed

    Chiocchetti, A G; Haslinger, D; Stein, J L; de la Torre-Ubieta, L; Cocchi, E; Rothämel, T; Lindlar, S; Waltes, R; Fulda, S; Geschwind, D H; Freitag, C M

    2016-01-01

    Genes for autism spectrum disorders (ASDs) are also implicated in fragile X syndrome (FXS), intellectual disabilities (ID) or schizophrenia (SCZ), and converge on neuronal function and differentiation. The SH-SY5Y neuroblastoma cell line, the most widely used system to study neurodevelopment, is currently discussed for its applicability to model cortical development. We implemented an optimal neuronal differentiation protocol of this system and evaluated neurodevelopment at the transcriptomic level using the CoNTeXT framework, a machine-learning algorithm based on human post-mortem brain data estimating developmental stage and regional identity of transcriptomic signatures. Our improved model in contrast to currently used SH-SY5Y models does capture early neurodevelopmental processes with high fidelity. We applied regression modelling, dynamic time warping analysis, parallel independent component analysis and weighted gene co-expression network analysis to identify activated gene sets and networks. Finally, we tested and compared these sets for enrichment of risk genes for neuropsychiatric disorders. We confirm a significant overlap of genes implicated in ASD with FXS, ID and SCZ. However, counterintuitive to this observation, we report that risk genes affect pathways specific for each disorder during early neurodevelopment. Genes implicated in ASD, ID, FXS and SCZ were enriched among the positive regulators, but only ID-implicated genes were also negative regulators of neuronal differentiation. ASD and ID genes were involved in dendritic branching modules, but only ASD risk genes were implicated in histone modification or axonal guidance. Only ID genes were over-represented among cell cycle modules. We conclude that the underlying signatures are disorder-specific and that the shared genetic architecture results in overlaps across disorders such as ID in ASD. Thus, adding developmental network context to genetic analyses will aid differentiating the pathophysiology

  14. Opsin expression in Limulus eyes: a UV opsin is expressed in each eye type and co-expressed with a visible light-sensitive opsin in ventral larval eyes

    PubMed Central

    Battelle, Barbara-Anne; Kempler, Karen E.; Harrison, Alexandra; Dugger, Donald R.; Payne, Richard

    2014-01-01

    The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors. PMID:24948643

  15. Opsin expression in Limulus eyes: a UV opsin is expressed in each eye type and co-expressed with a visible light-sensitive opsin in ventral larval eyes.

    PubMed

    Battelle, Barbara-Anne; Kempler, Karen E; Harrison, Alexandra; Dugger, Donald R; Payne, Richard

    2014-09-01

    The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors. PMID:24948643

  16. Substantially elevating the levels of αB-crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation

    PubMed Central

    Xu, Guilian; Fromholt, Susan; Ayers, Jacob I.; Brown, Hilda; Siemienski, Zoe; Crosby, Keith W.; Mayer, Christopher A.; Janus, Christopher; Borchelt, David R.

    2015-01-01

    There has been great interest in enhancing endogenous protein maintenance pathways such as the heat-shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in ALS and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co-expression of αB-crystallin (αB-crys) has been shown to inhibit the formation of detergent-insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB-crys at >6-fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB-crys alone contained 20% more motor neurons per section than littermate controls. Raising αB-crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent-insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB-crys in spinal motor neurons by 6-fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation. PMID:25557022

  17. Characterization of claustral neurons by comparative gene expression profiling and dye-injection analyses.

    PubMed

    Watakabe, Akiya; Ohsawa, Sonoko; Ichinohe, Noritaka; Rockland, Kathleen S; Yamamori, Tetsuo

    2014-01-01

    The identity of the claustrum as a part of cerebral cortex, and in particular of the adjacent insular cortex, has been investigated by connectivity features and patterns of gene expression. In the present paper, we mapped the cortical and claustral expression of several cortical genes in rodent and macaque monkey brains (nurr1, latexin, cux2, and netrinG2) to further assess shared features between cortex and claustrum. In mice, these genes were densely expressed in the claustrum, but very sparsely in the cortex and not present in the striatum. To test whether the cortical vs. claustral cell types can be distinguished by co-expression of these genes, we performed a panel of double ISH in mouse and macaque brain. NetrinG2 and nurr1 genes were co-expressed across entire cortex and claustrum, but cux2 and nurr1 were co-expressed only in the insular cortex and claustrum. Latexin was expressed, in the macaque, only in the claustrum. The nurr1 (+) claustral neurons expressed VGluT1, a marker for cortical glutamatergic cells and send cortical projections. Taken together, our data suggest a partial commonality between claustral neurons and a subtype of cortical neurons in the monkey brain. Moreover, in the embryonic (E110) macaque brain, many nurr1 (+) neurons were scattered in the white matter between the claustrum and the insular cortex, possibly representing their migratory history. In a second set of experiments, we injected Lucifer Yellow intracellularly in mouse and rat slices to investigate whether dendrites of insular and claustral neurons can cross the border of the two brain regions. Dendrites of claustral neurons did not invade the overlying insular territory. In summary, gene expression profile of the claustrum is similar to that of the neocortex, in both rodent and macaque brains, but with modifications in density of expression and cellular co-localization of specific genes. PMID:24904319

  18. Neurochemical characterization of pERK-expressing spinal neurons in histamine-induced itch

    PubMed Central

    Jiang, Guan-Yu; Dai, Meng-Han; Huang, Kun; Chai, Guo-Dong; Chen, Jia-Yin; Chen, Ling; Lang, Bing; Wang, Qing-Xiu; St Clair, David; McCaig, Colin; Ding, Yu-Qiang; Zhang, Ling

    2015-01-01

    Acute itch is divided into histamine- and non-histamine-dependent subtypes, and our previous study has shown that activation of ERK signaling in the spinal dorsal horn (SDH) is required selectively for histamine-induced itch sensation. Morphological characteristics of pERK-expressing neurons are required for exploring the mechanism underlying spinal itch sensation. To investigate whether pERK-expressing neurons are supraspinally-projecting neurons, we injected Fluorogold (FG) into the ventrobasal thalamic complex (VB) and parabrachial region, the two major spinal ascending sites in rodents. A small number (1%) of pERK-positive neurons were labeled by FG, suggesting that histamine-induced activation of ERK is primarily located in local SDH neurons. We then examined the co-localization of pERK with Calbindin and Lmx1b, which are expressed by excitatory neurons, and found that more than half (58%) of pERK-positive neurons expressed Lmx1b, but no co-expression with Calbindin was observed. On the other hand, approximately 7% of pERK-positive neurons expressed GAD67, and 27% of them contained Pax2. These results support the idea that pERK-expressing neurons serve as a component of local neuronal circuits for processing itch sensation in the spinal cord. PMID:26248539

  19. Protein Co-Expression Analysis as a Strategy to Complement a Standard Quantitative Proteomics Approach: Case of a Glioblastoma Multiforme Study

    PubMed Central

    Deighton, Ruth F.

    2016-01-01

    Although correlation network studies from co-expression analysis are increasingly popular, they are rarely applied to proteomics datasets. Protein co-expression analysis provides a complementary view of underlying trends, which can be overlooked by conventional data analysis. The core of the present study is based on Weighted Gene Co-expression Network Analysis applied to a glioblastoma multiforme proteomic dataset. Using this method, we have identified three main modules which are associated with three different membrane associated groups; mitochondrial, endoplasmic reticulum, and a vesicle fraction. The three networks based on protein co-expression were assessed against a publicly available database (STRING) and show a statistically significant overlap. Each of the three main modules were de-clustered into smaller networks using different strategies based on the identification of highly connected networks, hierarchical clustering and enrichment of Gene Ontology functional terms. Most of the highly connected proteins found in the endoplasmic reticulum module were associated with redox activity while a core of the unfolded protein response was identified in addition to proteins involved in oxidative stress pathways. The proteins composing the electron transfer chain were found differently affected with proteins from mitochondrial Complex I being more down-regulated than proteins from Complex III. Finally, the two pyruvate kinases isoforms show major differences in their co-expressed protein networks suggesting roles in different cellular locations. PMID:27571357

  20. Protein Co-Expression Analysis as a Strategy to Complement a Standard Quantitative Proteomics Approach: Case of a Glioblastoma Multiforme Study.

    PubMed

    Kanonidis, Evangelos I; Roy, Marcia M; Deighton, Ruth F; Le Bihan, Thierry

    2016-01-01

    Although correlation network studies from co-expression analysis are increasingly popular, they are rarely applied to proteomics datasets. Protein co-expression analysis provides a complementary view of underlying trends, which can be overlooked by conventional data analysis. The core of the present study is based on Weighted Gene Co-expression Network Analysis applied to a glioblastoma multiforme proteomic dataset. Using this method, we have identified three main modules which are associated with three different membrane associated groups; mitochondrial, endoplasmic reticulum, and a vesicle fraction. The three networks based on protein co-expression were assessed against a publicly available database (STRING) and show a statistically significant overlap. Each of the three main modules were de-clustered into smaller networks using different strategies based on the identification of highly connected networks, hierarchical clustering and enrichment of Gene Ontology functional terms. Most of the highly connected proteins found in the endoplasmic reticulum module were associated with redox activity while a core of the unfolded protein response was identified in addition to proteins involved in oxidative stress pathways. The proteins composing the electron transfer chain were found differently affected with proteins from mitochondrial Complex I being more down-regulated than proteins from Complex III. Finally, the two pyruvate kinases isoforms show major differences in their co-expressed protein networks suggesting roles in different cellular locations. PMID:27571357

  1. A fiber-modified adenovirus co-expressing HSV-TK and Coli.NTR enhances antitumor activities in breast cancer cells

    PubMed Central

    Zhan, Yang; Yu, Bin; Wang, Zhen; Zhang, Yu; Zhang, Hai-Hong; Wu, Hao; Feng, Xiao; Geng, Ran-Shen; Kong, Wei; Yu, Xiang-Hui

    2014-01-01

    Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer. PMID:25031704

  2. Expression of early growth response protein 1 in vasopressin neurones of the rat anterior olfactory nucleus following social odour exposure

    PubMed Central

    Wacker, Douglas W; Tobin, Vicky A; Noack, Julia; Bishop, Valerie R; Duszkiewicz, Adrian J; Engelmann, Mario; Meddle, Simone L; Ludwig, Mike

    2010-01-01

    The anterior olfactory nucleus (AON), a component of the main olfactory system, is a cortical region that processes olfactory information and acts as a relay between the main olfactory bulbs and higher brain regions such as the piriform cortex. Utilizing a transgenic rat in which an enhanced green fluorescent protein reporter gene is expressed in vasopressin neurones (eGFP-vasopressin), we have discovered a population of vasopressin neurones in the AON. These vasopressin neurones co-express vasopressin V1 receptors. They also co-express GABA and calbinin-D28k indicating that they are neurochemically different from the newly described vasopressin neurons in the main olfactory bulb. We utilized the immediate early gene product, early growth response protein 1 (Egr-1), to examine the functional role of these vasopressin neurons in processing social and non-social odours in the AON. Exposure of adult rats to a conspecific juvenile or a heterospecific predator odour leads to increases in Egr-1 expression in the AON in a subregion specific manner. However, only exposure to a juvenile increases Egr-1 expression in AON vasopressin neurons. These data suggest that vasopressin neurones in the AON may be selectively involved in the coding of social odour information. PMID:20921194

  3. Use of transcriptomics and co-expression networks to analyze the interconnections between nitrogen assimilation and photorespiratory metabolism

    PubMed Central

    Pérez-Delgado, Carmen M.; Moyano, Tomás C.; García-Calderón, Margarita; Canales, Javier; Gutiérrez, Rodrigo A.; Márquez, Antonio J.; Betti, Marco

    2016-01-01

    Nitrogen is one of the most important nutrients for plants and, in natural soils, its availability is often a major limiting factor for plant growth. Here we examine the effect of different forms of nitrogen nutrition and of photorespiration on gene expression in the model legume Lotus japonicus with the aim of identifying regulatory candidate genes co-ordinating primary nitrogen assimilation and photorespiration. The transcriptomic changes produced by the use of different nitrogen sources in leaves of L. japonicus plants combined with the transcriptomic changes produced in the same tissue by different photorespiratory conditions were examined. The results obtained provide novel information on the possible role of plastidic glutamine synthetase in the response to different nitrogen sources and in the C/N balance of L. japonicus plants. The use of gene co-expression networks establishes a clear relationship between photorespiration and primary nitrogen assimilation and identifies possible transcription factors connected to the genes of both routes. PMID:27117340

  4. Use of transcriptomics and co-expression networks to analyze the interconnections between nitrogen assimilation and photorespiratory metabolism.

    PubMed

    Pérez-Delgado, Carmen M; Moyano, Tomás C; García-Calderón, Margarita; Canales, Javier; Gutiérrez, Rodrigo A; Márquez, Antonio J; Betti, Marco

    2016-05-01

    Nitrogen is one of the most important nutrients for plants and, in natural soils, its availability is often a major limiting factor for plant growth. Here we examine the effect of different forms of nitrogen nutrition and of photorespiration on gene expression in the model legume Lotus japonicus with the aim of identifying regulatory candidate genes co-ordinating primary nitrogen assimilation and photorespiration. The transcriptomic changes produced by the use of different nitrogen sources in leaves of L. japonicus plants combined with the transcriptomic changes produced in the same tissue by different photorespiratory conditions were examined. The results obtained provide novel information on the possible role of plastidic glutamine synthetase in the response to different nitrogen sources and in the C/N balance of L. japonicus plants. The use of gene co-expression networks establishes a clear relationship between photorespiration and primary nitrogen assimilation and identifies possible transcription factors connected to the genes of both routes. PMID:27117340

  5. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli

    PubMed Central

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V.; Patel, Bhumit A.; Yeh, Syun-Ru; Rousseau, Dennis L.; Crane, Brian R.

    2010-01-01

    Over-expression of heme binding proteins in E. coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His- ligated hemes. PMID:20303407

  6. Assessing the Biological Significance of Gene Expression Signatures and Co-Expression Modules by Studying Their Network Properties

    PubMed Central

    Minguez, Pablo; Dopazo, Joaquin

    2011-01-01

    Microarray experiments have been extensively used to define signatures, which are sets of genes that can be considered markers of experimental conditions (typically diseases). Paradoxically, in spite of the apparent functional role that might be attributed to such gene sets, signatures do not seem to be reproducible across experiments. Given the close relationship between function and protein interaction, network properties can be used to study to what extent signatures are composed of genes whose resulting proteins show a considerable level of interaction (and consequently a putative common functional role). We have analysed 618 signatures and 507 modules of co-expression in cancer looking for significant values of four main protein-protein interaction (PPI) network parameters: connection degree, cluster coefficient, betweenness and number of components. A total of 3904 gene ontology (GO) modules, 146 KEGG pathways, and 263 Biocarta pathways have been used as functional modules of reference. Co-expression modules found in microarray experiments display a high level of connectivity, similar to the one shown by conventional modules based on functional definitions (GO, KEGG and Biocarta). A general observation for all the classes studied is that the networks formed by the modules improve their topological parameters when an external protein is allowed to be introduced within the paths (up to the 70% of GO modules show network parameters beyond the random expectation). This fact suggests that functional definitions are incomplete and some genes might still be missing. Conversely, signatures are clearly not capturing the altered functions in the corresponding studies. This is probably because the way in which the genes have been selected in the signatures is too conservative. These results suggest that gene selection methods which take into account relationships among genes should be superior to methods that assume independence among genes outside their functional

  7. Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse.

    PubMed

    Kurniasih, Sari Dewi; Alfi, Almasul; Natalia, Dessy; Radjasa, Ocky Karna; Nurachman, Zeily

    2014-01-01

    At least a combination of endoglucanase (EglII) and β-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed β-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase. PMID:24598011

  8. Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival

    PubMed Central

    Kong, Yink Heay; Syed Zanaruddin, Sharifah Nurain; Lau, Shin Hin; Ramanathan, Anand; Kallarakkal, Thomas George; Vincent-Chong, Vui King; Wan Mustafa, Wan Mahadzir; Abraham, Mannil Thomas; Abdul Rahman, Zainal Ariff; Zain, Rosnah Binti; Cheong, Sok Ching

    2015-01-01

    Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer’s aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder’s grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management. PMID:26214683

  9. Improved 1, 2, 4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins.

    PubMed

    Lu, Xinyao; He, Shuying; Zong, Hong; Song, Jian; Chen, Wen; Zhuge, Bin

    2016-09-01

    1, 2, 4-Butanetriol (BT) is a high-value non-natural chemical and has important applications in polymers, medical production and military industry. In the constructed BT biosynthesis pathway from xylose in Escherichia coli, the xylose dehydrogenase (Xdh) and the benzoylformate decarboxylase (MdlC) are heterologous enzymes and the activity of MdlC is the key limiting factor for BT production. In this study, six chaperone protein systems were introduced into the engineered E. coli harboring the recombinant BT pathway. The chaperone GroES-GroEL was beneficial to Xdh activity but had a negative effect on MdlC activity and BT titer. The plasmid pTf16 containing the tig gene (trigger factor) was beneficial to Xdh and MdlC activities and improved the BT titer from 0.42 to 0.56 g/l from 20 g/l xylose. However, co-expression of trigger factor and GroES-GroEL simultaneously reduced the activity of MdlC and had no effect on the BT production. The plasmid pKJE7 harboring dnaK-dnaJ-grpE showed significant negative effects on these enzyme activities and cell growth, leading to completely restrained the BT production. Similarly, co-expression of DnaKJ-GrpPE and GroES-GroEL simultaneously reduced Xdh and MdlC activities and decreased the BT titer by 45.2 %. The BT production of the engineered E. coli harboring pTf16 was further improved to the highest level at 1.01 g/l under pH control (pH 7). This work showed the potential application of chaperone proteins in microorganism engineering to get high production of target compounds as an effective and valuable tool. PMID:27430516

  10. Gene Co-Expression Network Analysis Provides Novel Insights into Myostatin Regulation at Three Different Mouse Developmental Timepoints

    PubMed Central

    Yang, Xuerong; Koltes, James E.; Park, Carissa A.; Chen, Daiwen; Reecy, James M.

    2015-01-01

    Myostatin (Mstn) knockout mice exhibit large increases in skeletal muscle mass. However, relatively few of the genes that mediate or modify MSTN effects are known. In this study, we performed co-expression network analysis using whole transcriptome microarray data from MSTN-null and wild-type mice to identify genes involved in important biological processes and pathways related to skeletal muscle and adipose development. Genes differentially expressed between wild-type and MSTN-null mice were further analyzed for shared DNA motifs using DREME. Differentially expressed genes were identified at 13.5 d.p.c. during primary myogenesis and at d35 during postnatal muscle development, but not at 17.5 d.p.c. during secondary myogenesis. In total, 283 and 2034 genes were differentially expressed at 13.5 d.p.c. and d35, respectively. Over-represented transcription factor binding sites in differentially expressed genes included SMAD3, SP1, ZFP187, and PLAGL1. The use of regulatory (RIF) and phenotypic (PIF) impact factor and differential hubbing co-expression analyses identified both known and potentially novel regulators of skeletal muscle growth, including Apobec2, Atp2a2, and Mmp13 at d35 and Sox2, Tmsb4x, and Vdac1 at 13.5 d.p.c. Among the genes with the highest PIF scores were many fiber type specifying genes. The use of RIF, PIF, and differential hubbing analyses identified both known and potentially novel regulators of muscle development. These results provide new details of how MSTN may mediate transcriptional regulation as well as insight into novel regulators of MSTN signal transduction that merit further study regarding their physiological roles in muscle and adipose development. PMID:25695797

  11. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    PubMed

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect. PMID:25479595

  12. City block distance and rough-fuzzy clustering for identification of co-expressed microRNAs.

    PubMed

    Paul, Sushmita; Maji, Pradipta

    2014-06-01

    The microRNAs or miRNAs are short, endogenous RNAs having ability to regulate mRNA expression at the post-transcriptional level. Various studies have revealed that miRNAs tend to cluster on chromosomes. The members of a cluster that are in close proximity on chromosomes are highly likely to be processed as co-transcribed units. Therefore, a large proportion of miRNAs are co-expressed. Expression profiling of miRNAs generates a huge volume of data. Complicated networks of miRNA-mRNA interaction increase the challenges of comprehending and interpreting the resulting mass of data. In this regard, this paper presents a clustering algorithm in order to extract meaningful information from miRNA expression data. It judiciously integrates the merits of rough sets, fuzzy sets, the c-means algorithm, and the normalized range-normalized city block distance to discover co-expressed miRNA clusters. While the membership functions of fuzzy sets enable efficient handling of overlapping partitions in a noisy environment, the concept of lower and upper approximations of rough sets deals with uncertainty, vagueness, and incompleteness in cluster definition. The city block distance is used to compute the membership functions of fuzzy sets and to find initial partition of a data set, and therefore helps to handle minute differences between two miRNA expression profiles. The effectiveness of the proposed approach, along with a comparison with other related methods, is demonstrated for several miRNA expression data sets using different cluster validity indices. Moreover, the gene ontology is used to analyze the functional consistency and biological significance of generated miRNA clusters. PMID:24682049

  13. Origin of Co-Expression Patterns in E.coli and S.cerevisiae Emerging from Reverse Engineering Algorithms

    PubMed Central

    Zampieri, Mattia; Soranzo, Nicola; Bianchini, Daniele; Altafini, Claudio

    2008-01-01

    Background The concept of reverse engineering a gene network, i.e., of inferring a genome-wide graph of putative gene-gene interactions from compendia of high throughput microarray data has been extensively used in the last few years to deduce/integrate/validate various types of “physical” networks of interactions among genes or gene products. Results This paper gives a comprehensive overview of which of these networks emerge significantly when reverse engineering large collections of gene expression data for two model organisms, E.coli and S.cerevisiae, without any prior information. For the first organism the pattern of co-expression is shown to reflect in fine detail both the operonal structure of the DNA and the regulatory effects exerted by the gene products when co-participating in a protein complex. For the second organism we find that direct transcriptional control (e.g., transcription factor–binding site interactions) has little statistical significance in comparison to the other regulatory mechanisms (such as co-sharing a protein complex, co-localization on a metabolic pathway or compartment), which are however resolved at a lower level of detail than in E.coli. Conclusion The gene co-expression patterns deduced from compendia of profiling experiments tend to unveil functional categories that are mainly associated to stable bindings rather than transient interactions. The inference power of this systematic analysis is substantially reduced when passing from E.coli to S.cerevisiae. This extensive analysis provides a way to describe the different complexity between the two organisms and discusses the critical limitations affecting this type of methodologies. PMID:18714358

  14. Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

    PubMed Central

    Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

    2008-01-01

    Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

  15. Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications.

    PubMed

    Harb, Kawssar; Magrinelli, Elia; Nicolas, Céline S; Lukianets, Nikita; Frangeul, Laura; Pietri, Mariel; Sun, Tao; Sandoz, Guillaume; Grammont, Franck; Jabaudon, Denis; Studer, Michele; Alfano, Christian

    2016-01-01

    During cortical development, the identity of major classes of long-distance projection neurons is established by the expression of molecular determinants, which become gradually restricted and mutually exclusive. However, the mechanisms by which projection neurons acquire their final properties during postnatal stages are still poorly understood. In this study, we show that the number of neurons co-expressing Ctip2 and Satb2, respectively involved in the early specification of subcerebral and callosal projection neurons, progressively increases after birth in the somatosensory cortex. Ctip2/Satb2 postnatal co-localization defines two distinct neuronal subclasses projecting either to the contralateral cortex or to the brainstem suggesting that Ctip2/Satb2 co-expression may refine their properties rather than determine their identity. Gain- and loss-of-function approaches reveal that the transcriptional adaptor Lmo4 drives this maturation program through modulation of epigenetic mechanisms in a time- and area-specific manner, thereby indicating that a previously unknown genetic program postnatally promotes the acquisition of final subtype-specific features. PMID:26814051

  16. Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications

    PubMed Central

    Harb, Kawssar; Magrinelli, Elia; Nicolas, Céline S; Lukianets, Nikita; Frangeul, Laura; Pietri, Mariel; Sun, Tao; Sandoz, Guillaume; Grammont, Franck; Jabaudon, Denis; Studer, Michèle; Alfano, Christian

    2016-01-01

    During cortical development, the identity of major classes of long-distance projection neurons is established by the expression of molecular determinants, which become gradually restricted and mutually exclusive. However, the mechanisms by which projection neurons acquire their final properties during postnatal stages are still poorly understood. In this study, we show that the number of neurons co-expressing Ctip2 and Satb2, respectively involved in the early specification of subcerebral and callosal projection neurons, progressively increases after birth in the somatosensory cortex. Ctip2/Satb2 postnatal co-localization defines two distinct neuronal subclasses projecting either to the contralateral cortex or to the brainstem suggesting that Ctip2/Satb2 co-expression may refine their properties rather than determine their identity. Gain- and loss-of-function approaches reveal that the transcriptional adaptor Lmo4 drives this maturation program through modulation of epigenetic mechanisms in a time- and area-specific manner, thereby indicating that a previously unknown genetic program postnatally promotes the acquisition of final subtype-specific features. DOI: http://dx.doi.org/10.7554/eLife.09531.001 PMID:26814051

  17. Brain Neurons as Quantum Computers:

    NASA Astrophysics Data System (ADS)

    Bershadskii, A.; Dremencov, E.; Bershadskii, J.; Yadid, G.

    The question: whether quantum coherent states can sustain decoherence, heating and dissipation over time scales comparable to the dynamical timescales of brain neurons, has been actively discussed in the last years. A positive answer on this question is crucial, in particular, for consideration of brain neurons as quantum computers. This discussion was mainly based on theoretical arguments. In the present paper nonlinear statistical properties of the Ventral Tegmental Area (VTA) of genetically depressive limbic brain are studied in vivo on the Flinders Sensitive Line of rats (FSL). VTA plays a key role in the generation of pleasure and in the development of psychological drug addiction. We found that the FSL VTA (dopaminergic) neuron signals exhibit multifractal properties for interspike frequencies on the scales where healthy VTA dopaminergic neurons exhibit bursting activity. For high moments the observed multifractal (generalized dimensions) spectrum coincides with the generalized dimensions spectrum calculated for a spectral measure of a quantum system (so-called kicked Harper model, actively used as a model of quantum chaos). This observation can be considered as a first experimental (in vivo) indication in the favor of the quantum (at least partially) nature of brain neurons activity.

  18. Midline thalamic neurons are differentially engaged during hippocampus network oscillations.

    PubMed

    Lara-Vásquez, Ariel; Espinosa, Nelson; Durán, Ernesto; Stockle, Marcelo; Fuentealba, Pablo

    2016-01-01

    The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits. PMID:27411890

  19. Midline thalamic neurons are differentially engaged during hippocampus network oscillations

    PubMed Central

    Lara-Vásquez, Ariel; Espinosa, Nelson; Durán, Ernesto; Stockle, Marcelo; Fuentealba, Pablo

    2016-01-01

    The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits. PMID:27411890

  20. Ethanol and neuronal metabolism.

    PubMed

    Mandel, P; Ledig, M; M'Paria, J R

    1980-01-01

    The effect of ethanol on membrane enzymes (Na+, K+ and Mg2+ ATPases, 5'-nucleotidase, adenylate cyclase) alcohol dehydrogenase, aldehyde dehydrogenase and superoxide dismutase were studied in nerve cells (established cell lines, primary cultures of chick and rat brain) cultured in the presence of 100 mM ethanol, and in total rat brain, following various ethanol treatments of the rats (20% ethanol as the sole liquid source, intraperitoneal injection). The results show a difference between neuronal and glial cells. Most of the observed changes in enzymatic activities returned rapidly to control values when ethanol was withdrawn from the culture medium or from the diet. Alcohol dehydrogenase was more stimulated by ethanol than aldehyde dehydrogenase; therefore acetaldehyde may be accumulated. The inhibition of superoxide dismutase activity may allow an accumulation of cytotoxic O2- radicals in nervous tissue and may explain the polymorphism of lesions brought about by alcohol intoxication. PMID:6264495

  1. Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells

    PubMed Central

    Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

    2016-01-01

    This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

  2. Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells.

    PubMed

    Cheng, Yu-Che; Huang, Chi-Jung; Lee, Yih-Jing; Tien, Lu-Tai; Ku, Wei-Chi; Chien, Raymond; Lee, Fa-Kung; Chien, Chih-Cheng

    2016-01-01

    This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons. PMID:27444754

  3. CRF neurons in the ventral tegmental area control the aversive effects of nicotine withdrawal and promote escalation of nicotine intake

    PubMed Central

    Grieder, Taryn E.; Herman, Melissa A.; Contet, Candice; Tan, Laura A.; Vargas-Perez, Hector; Cohen, Ami; Chwalek, Michal; Maal-Bared, Geith; Freiling, John; Schlosburg, Joel E; Clarke, Laura; Crawford, Elena; Koebel, Pascale; Canonigo, Vez; Sanna, Pietro; Tapper, Andrew; Roberto, Marisa; Kieffer, Brigitte L.; Sawchenko, Paul E.; Koob, George F.; van der Kooy, Derek; George, Olivier

    2014-01-01

    SUMMARY Dopaminergic neurons in the ventral tegmental area (VTA) are well known for their role in mediating the positive reinforcing effects of drugs of abuse. Here, we identify in rodents and humans a population of VTA dopamine neurons co-expressing corticotropin releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates CRF mRNA in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors, and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of CRF mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal, and limited the escalation of nicotine intake. These results link the brain reward and stress systems within the same brain region in signaling the negative motivational effects of nicotine withdrawal. PMID:25402857

  4. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

    PubMed Central

    2014-01-01

    Background Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic

  5. Serotonin-2C and -2A Receptor Co-expression on Cells in the Rat Medial Prefrontal Cortex

    PubMed Central

    Nocjar, Christine; Alex, Katherine D; Sonneborn, Alex; Abbas, Atheir I; Roth, Bryan L; Pehek, Elizabeth A

    2015-01-01

    Neural function within the medial prefrontal cortex (mPFC) regulates normal cognition, attention and impulse control, implicating neuroregulatory abnormalities within this region in mental dysfunction related to schizophrenia, depression and drug abuse. Both serotonin -2A (5-HT2A) and -2C (5-HT2C) receptors are known to be important in neuropsychiatric drug action and are distributed throughout the mPFC. However, their interactive role in serotonergic cortical regulation is poorly understood. While the main signal transduction mechanism for both receptors is stimulation of phosphoinositide production, they can have opposite effects downstream. 5-HT2A versus 5-HT2C receptor activation oppositely regulates behavior and can oppositely affect neurochemical release within the mPFC. These distinct receptor effects could be caused by their differential cellular distribution within the cortex and/or other areas. It is known that both receptors are located on GABAergic and pyramidal cells within the mPFC, but it is not clear whether they are expressed on the same or different cells. The present work employed immunofluorescence with confocal microscopy to examine this in layers V-VI of the prelimbic mPFC. The majority of GABA cells in the deep prelimbic mPFC expressed 5-HT2C receptor immunoreactivity. Furthermore, most cells expressing 5-HT2C receptor immunoreactivity notably co-expressed 5-HT2A receptors. However, 27% of 5-HT2C receptor immunoreactive cells were not GABAergic, indicating that a population of prelimbic pyramidal projection cells could express the 5-HT2C receptor. Indeed, some cells with 5-HT2C and 5-HT2A receptor co-labeling had a pyramidal shape and were expressed in the typical layered fashion of pyramidal cells. This indirectly demonstrates that 5-HT2C and 5-HT2A receptors may be commonly co-expressed on GABAergic cells within the deep layers of the prelimbic mPFC and perhaps co-localized on a small population of local pyramidal projection cells. Thus a

  6. ATTED-II: a database of co-expressed genes and cis elements for identifying co-regulated gene groups in Arabidopsis

    PubMed Central

    Obayashi, Takeshi; Kinoshita, Kengo; Nakai, Kenta; Shibaoka, Masayuki; Hayashi, Shinpei; Saeki, Motoshi; Shibata, Daisuke; Saito, Kazuki; Ohta, Hiroyuki

    2007-01-01

    Publicly available database of co-expressed gene sets would be a valuable tool for a wide variety of experimental designs, including targeting of genes for functional identification or for regulatory investigation. Here, we report the construction of an Arabidopsis thaliana trans-factor and cis-element prediction database (ATTED-II) that provides co-regulated gene relationships based on co-expressed genes deduced from microarray data and the predicted cis elements. ATTED-II () includes the following features: (i) lists and networks of co-expressed genes calculated from 58 publicly available experimental series, which are composed of 1388 GeneChip data in A.thaliana; (ii) prediction of cis-regulatory elements in the 200 bp region upstream of the transcription start site to predict co-regulated genes amongst the co-expressed genes; and (iii) visual representation of expression patterns for individual genes. ATTED-II can thus help researchers to clarify the function and regulation of particular genes and gene networks. PMID:17130150

  7. Correlated mRNAs and miRNAs from co-expression and regulatory networks affect porcine muscle and finally meat properties

    PubMed Central

    2013-01-01

    Background Physiological processes aiding the conversion of muscle to meat involve many genes associated with muscle structure and metabolic processes. MicroRNAs regulate networks of genes to orchestrate cellular functions, in turn regulating phenotypes. Results We applied weighted gene co-expression network analysis to identify co-expression modules that correlated to meat quality phenotypes and were highly enriched for genes involved in glucose metabolism, response to wounding, mitochondrial ribosome, mitochondrion, and extracellular matrix. Negative correlation of miRNA with mRNA and target prediction were used to select transcripts out of the modules of trait-associated mRNAs to further identify those genes that are correlated with post mortem traits. Conclusions Porcine muscle co-expression transcript networks that correlated to post mortem traits were identified. The integration of miRNA and mRNA expression analyses, as well as network analysis, enabled us to interpret the differentially-regulated genes from a systems perspective. Linking co-expression networks of transcripts and hierarchically organized pairs of miRNAs and mRNAs to meat properties yields new insight into several biological pathways underlying phenotype differences. These pathways may also be diagnostic for many myopathies, which are accompanied by deficient nutrient and oxygen supply of muscle fibers. PMID:23915301

  8. Visual Display and Co-Expressivity as Students Strive for Intersubjectivity in a Spatial Reasoning Task. WCER Working Paper No. 2005-10

    ERIC Educational Resources Information Center

    Nathan, Mitchell J.; Eilam, Billie

    2005-01-01

    This paper explores the role of co-expressivity of drawing, speech, and gesture as sixth graders strive for intersubjectivity in their attempts to communicate solutions of a spatial reasoning task. Several obstacles to intersubjectivity are documented, particularly conflicts between a literal and geometric interpretation, limits of students'…

  9. Co-Expression of Bmi-1 and Podoplanin Predicts Overall Survival in Patients With Squamous Cell Carcinoma of the Head and Neck Treated With Radio(chemo)therapy

    SciTech Connect

    Vormittag, Laurenz; Thurnher, Dietmar; Geleff, Silvana; Pammer, Johannes; Heiduschka, Gregor; Brunner, Markus; Grasl, Matthaeus Ch.; Erovic, Boban M.

    2009-03-01

    Purpose: This study was conducted to determine the expression of Bmi-1 and podoplanin in healthy oral mucosa and in untreated tumor tissues samples of patients with squamous cell carcinomas of the head and neck. All patients were treated by primary radio(chemo)therapy. Methods and Materials: The expression of Bmi-1 and podoplanin was immunohistochemically evaluated in 12 normal oral mucosa and 63 tumor specimens and correlated with patients' clinical data. Results: In healthy mucosa expression of Bmi-1 and podoplanin was restricted to the basal cell layer. Expression of both proteins was found in 79% and 86% of our tumor samples, respectively. In 17 and 8 samples, Bmi-1 and podoplanin were co-expressed at the invasive border or diffuse in the bulk of the tumor, respectively. Univariate analysis showed that the co-expression of Bmi-1 and podoplanin correlated to decreased overall survival (p = 0.044). Moreover, multivariate testing identified high expression of podoplanin (p = 0.044), co-expression of Bmi-1 and podoplanin (p = 0.007) and lack of response to therapy (p < 0.0001) as predictors of shortened overall survival in patients treated with primary radio(chemo)therapy. Conclusions: Bmi-1 and podoplanin are expressed at the invasive front of squamous cell carcinomas of the head and neck. Co-expression of Bmi-1 and podoplanin predicts significantly overall survival of patients treated with primary radio(chemo)therapy.

  10. Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF164 in skeletal muscle

    PubMed Central

    Gianni-Barrera, Roberto; Burger, Maximilian; Wolff, Thomas; Heberer, Michael; Schaefer, Dirk J.; Gürke, Lorenz; Mujagic, Edin; Banfi, Andrea

    2016-01-01

    Therapeutic angiogenesis by growth factor delivery is an attractive treatment strategy for ischemic diseases, yet clinical efficacy has been elusive. The angiogenic master regulator VEGF-A can induce aberrant angiogenesis if expressed above a threshold level. Since VEGF remains localized in the matrix around expressing cells, homogeneous dose distribution in target tissues is required, which is challenging. We found that co-expression of the pericyte-recruiting factor PDGF-BB at a fixed ratio with VEGF from a single bicistronic vector ensured normal angiogenesis despite heterogeneous high VEGF levels. Taking advantage of a highly controlled gene delivery platform, based on monoclonal populations of transduced myoblasts, in which every cell stably produces the same amount of each factor, here we rigorously investigated a) the dose-dependent effects, and b) the long-term safety and stability of VEGF and PDGF-BB co-expression in skeletal muscle. PDGF-BB co-expression did not affect the normal angiogenesis by low and medium VEGF doses, but specifically prevented vascular tumors by high VEGF, yielding instead normal and mature capillary networks, accompanied by robust arteriole formation. Induced angiogenesis persisted unchanged up to 4 months, while no tumors appeared. Therefore, PDGF-BB co-expression is an attractive strategy to improve safety and efficacy of therapeutic angiogenesis by VEGF gene delivery. PMID:26882992

  11. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    PubMed

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants. PMID:26284097

  12. Transcriptional comparison of human induced and primary midbrain dopaminergic neurons

    PubMed Central

    Xia, Ninuo; Zhang, Pengbo; Fang, Fang; Wang, Zhengyuan; Rothstein, Megan; Angulo, Benjamin; Chiang, Rosaria; Taylor, James; Reijo Pera, Renee A.

    2016-01-01

    Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson’s disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson’s Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression, especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies, including in vitro modeling of PD. However, further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. PMID:26842779

  13. Co-expression networks revealed potential core lncRNAs in the triple-negative breast cancer.

    PubMed

    Yang, Fan; Liu, Ye-Huan; Dong, Si-Yang; Yao, Zhi-Han; Lv, Lin; Ma, Rui-Min; Dai, Xuan-Xuan; Wang, Jiao; Zhang, Xiao-Hua; Wang, Ou-Chen

    2016-10-15

    Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with unfavorable outcome. It is urgent to explore novel biomarkers and potential therapeutic targets in this malignancy. Increasing knowledge of long noncoding RNAs (lncRNAs) significantly deepens our understanding of cancer biology. Here, we sequenced eight paired TNBC tumor tissues and non-cancerous tissues, and validated significantly differentially expressed lncRNAs. Gene ontology (GO) and pathway analysis were used to investigate the function of differentially expressed mRNAs. Further, potential core lncRNAs in TNBC were identified by co-expression networks. Kaplan-Meier analysis also indicated that breast cancer patients with lower expression level of rhabdomyosarcoma 2 associated transcript (RMST), one of the potential core lncRNAs, had worse overall survival. To the best of our knowledge, it was the first report that RMST was involved in breast cancer. Our research provided a rich resource to the research community for further investigating lncRNAs functions and identifying lncRNAs with diagnostic and therapeutic potentials in TNBC. PMID:27380926

  14. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  15. A genome-wide cis-regulatory element discovery method based on promoter sequences and gene co-expression networks

    PubMed Central

    2013-01-01

    Background Deciphering cis-regulatory networks has become an attractive yet challenging task. This paper presents a simple method for cis-regulatory network discovery which aims to avoid some of the common problems of previous approaches. Results Using promoter sequences and gene expression profiles as input, rather than clustering the genes by the expression data, our method utilizes co-expression neighborhood information for each individual gene, thereby overcoming the disadvantages of current clustering based models which may miss specific information for individual genes. In addition, rather than using a motif database as an input, it implements a simple motif count table for each enumerated k-mer for each gene promoter sequence. Thus, it can be used for species where previous knowledge of cis-regulatory motifs is unknown and has the potential to discover new transcription factor binding sites. Applications on Saccharomyces cerevisiae and Arabidopsis have shown that our method has a good prediction accuracy and outperforms a phylogenetic footprinting approach. Furthermore, the top ranked gene-motif regulatory clusters are evidently functionally co-regulated, and the regulatory relationships between the motifs and the enriched biological functions can often be confirmed by literature. Conclusions Since this method is simple and gene-specific, it can be readily utilized for insufficiently studied species or flexibly used as an additional step or data source for previous transcription regulatory networks discovery models. PMID:23368633

  16. Co-expression of Dorsal and Rel2 Negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta

    PubMed Central

    Zhong, Xue; Rao, Xiang-Jun; Yi, Hui-Yu; Lin, Xin-Yu; Huang, Xiao-Hong; Yu, Xiao-Qiang

    2016-01-01

    Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. Here we report the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. We for the first time confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggesting that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. Our results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides. PMID:26847920

  17. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    SciTech Connect

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  18. Identification of hub genes of pneumocyte senescence induced by thoracic irradiation using weighted gene co-expression network analysis

    PubMed Central

    XING, YONGHUA; ZHANG, JUNLING; LU, LU; LI, DEGUAN; WANG, YUEYING; HUANG, SONG; LI, CHENGCHENG; ZHANG, ZHUBO; LI, JIANGUO; MENG, AIMIN

    2016-01-01

    Irradiation commonly causes pneumocyte senescence, which may lead to severe fatal lung injury characterized by pulmonary dysfunction and respiratory failure. However, the molecular mechanism underlying the induction of pneumocyte senescence by irradiation remains to be elucidated. In the present study, weighted gene co-expression network analysis (WGCNA) was used to screen for differentially expressed genes, and to identify the hub genes and gene modules, which may be critical for senescence. A total of 2,916 differentially expressed genes were identified between the senescence and non-senescence groups following thoracic irradiation. In total, 10 gene modules associated with cell senescence were detected, and six hub genes were identified, including B-cell scaffold protein with ankyrin repeats 1, translocase of outer mitochondrial membrane 70 homolog A, actin filament-associated protein 1, Cd84, Nuf2 and nuclear factor erythroid 2. These genes were markedly associated with cell proliferation, cell division and cell cycle arrest. The results of the present study demonstrated that WGCNA of microarray data may provide further insight into the molecular mechanism underlying pneumocyte senescence. PMID:26572216

  19. Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase.

    PubMed

    Ma, Jiangfeng; Gou, Dongmei; Liang, Liya; Liu, Rongming; Chen, Xu; Zhang, Changqing; Zhang, Jiuhua; Chen, Kequan; Jiang, Min

    2013-08-01

    Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD(+). To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD(+) ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l(-1) glucose and produced 14.08 g l(-1) succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased. PMID:23740313

  20. Methyl green and nitrotetrazolium blue chloride co-expression in colon tissue: A hyperspectral microscopic imaging analysis

    NASA Astrophysics Data System (ADS)

    Li, Qingli; Liu, Hongying; Wang, Yiting; Sun, Zhen; Guo, Fangmin; Zhu, Jianzhong

    2014-12-01

    Histological observation of dual-stained colon sections is usually performed by visual observation under a light microscope, or by viewing on a computer screen with the assistance of image processing software in both research and clinical settings. These traditional methods are usually not sufficient to reliably differentiate spatially overlapping chromogens generated by different dyes. Hyperspectral microscopic imaging technology offers a solution for these constraints as the hyperspectral microscopic images contain information that allows differentiation between spatially co-located chromogens with similar but different spectra. In this paper, a hyperspectral microscopic imaging (HMI) system is used to identify methyl green and nitrotetrazolium blue chloride in dual-stained colon sections. Hyperspectral microscopic images are captured and the normalized score algorithm is proposed to identify the stains and generate the co-expression results. Experimental results show that the proposed normalized score algorithm can generate more accurate co-localization results than the spectral angle mapper algorithm. The hyperspectral microscopic imaging technology can enhance the visualization of dual-stained colon sections, improve the contrast and legibility of each stain using their spectral signatures, which is helpful for pathologist performing histological analyses.

  1. Enhancement of heavy metal accumulation by tissue specific co-expression of iaaM and ACC deaminase genes in plants.

    PubMed

    Zhang, Yong; Zhao, Lihong; Wang, Yao; Yang, Baoyu; Chen, Shiyun

    2008-06-01

    1-Aminocyclopropane deaminase (ACC) and tryptophan monooxygenase are two enzymes involved in plant senescence-inhibiting and growth-promoting regulation, respectively. In this study, two binary vectors were constructed in which the Agrobacterium iaaM gene was under the transcriptional control of a xylem-specific glycine-rich protein promoter alone, or co-expressed with the bacterial ACC deaminase gene, which was driven by the constitutive CaMV 35S promoter. Transgenic petunia shoots co-expressing both genes were able to root on medium supplemented with 7.5 mg l(-1) CoCl2. When T1 transgenic tobacco plants were grown in sand supplemented with Cu2+ and Co2+, tissue specific co-expression of both iaaM and ACC deaminase genes showed faster growth with larger biomass with a more extensive root system, and accumulated a greater amount of heavy metals than the empty vector control plants. When T1 transgenic tobacco plants were grown in soil watered with different concentrations of CuSO4, xylem specific expression of the iaaM gene caused the accumulation of more Cu2+ than the empty vector control at lower CuSO4 concentrations, but showed severe toxic symptoms at concentration of 100 mg l(-1) CuSO4. T1 transgenic plants co-expressing both genes accumulated more heavy metals into the plant shoots and can tolerate CuSO4 at 150 mg l(-1). In addition, plants co-expressing these two genes can grow well in a complex contaminated soil containing both inorganic and organic pollutants, while the growth of the control plants was greatly inhibited. PMID:18471863

  2. Pharmacological rescue of hERG currents carried out by G604S and wide type hERG co-expression.

    PubMed

    Huo, Jianhua; Zhang, Aifeng; Guo, Xueyan; Qiang, Hua; Liu, Ping; Bai, Ling; Ma, Aiqun

    2016-09-01

    Mutations in human ether-a-go-go-related gene (hERG) can lead to type 2 long-QT syndrome (LQT2). The authors previously identified the hERG mutation G604S results in a loss of function and obviously decreased current amplitude and impaired channel protein trafficking when co-expressed with WT-hERG. The present study further investigates the biological and electrophysiological consequences of pharmacologic chaperones in HEK293 cells expressing G604S-hERG or co-expressing G604S-hERG and WT-hERG. It was found that a low temperature (27°C), thapsigargin, NS1643 and E-4031 fail to rescue the G604S mutation. Interestingly, only E-4031 treatment resulted in a significant increase in hERG currents in cells co-expressing G604S-hERG and WT-hERG, correspondingly more mature protein band at 155 kDa by Western blotting and an increased membrane staining by confocal microscopy. In addition, E-4031 treatment shifted the steady-state half maximal activation voltage (V1/2 ) of the inactivation curve by +8 mV in cells co-expressing G604S-hERG and WT-hERG. The present experimental results suggest that a G604S mutation is resistant to pharmacological rescue. E-4031 treatment resulted in a significant increase in hERG currents by promoting the hERG channel processing and trafficking in cells co-expressing G604S-hERG and WT-hERG. PMID:27199074

  3. Olfactory Receptor Neuron Dysfunction in Schizophrenia

    PubMed Central

    Turetsky, Bruce I; Hahn, Chang-Gyu; Arnold, Steven E; Moberg, Paul J

    2012-01-01

    Olfactory impairments are a common feature of schizophrenia. Impairments in odor detection and odor identification are present early in the course of illness and among those at risk for the disorder. These behavioral impairments have been linked to both physiological and anatomical abnormalities in the neural substrates subserving olfaction, including relatively peripheral elements of the olfactory system. The location of olfactory receptor neurons in the nasal epithelium allows noninvasive access to these neurons in living subjects. This offers a unique opportunity to directly assess neuronal integrity in vivo in patients. The peripheral olfactory receptor neuron response to odor stimulation was assessed in 21 schizophrenia patients and 18 healthy comparison subjects. The electroolfactogram, representing the electrical depolarization of the olfactory receptor neurons, was recording following stimulation with different doses and durations of hydrogen sulfide, a pure olfactory nerve stimulant. Schizophrenia patients had abnormally large depolarization responses following odor stimulation, independent of clinical symptomatology, antipsychotic medication dosage or smoking history. Although the precise pathophysiological mechanism is unknown, this olfactory receptor neuron abnormality is consistent with several lines of evidence suggesting altered proliferation or maturation of olfactory receptor neuron cell lineages in schizophrenia. It is also consistent with emerging evidence of disruptions of cyclic AMP-mediated intracellular signaling mechanisms, and may be a marker of these disruptions. It unambiguously demonstrates that neurophysiological disturbances in schizophrenia are not limited to cortical and subcortical structures, but rather include even the most peripheral sensory neurons. PMID:18754006

  4. Olfactory receptor neuron dysfunction in schizophrenia.

    PubMed

    Turetsky, Bruce I; Hahn, Chang-Gyu; Arnold, Steven E; Moberg, Paul J

    2009-02-01

    Olfactory impairments are a common feature of schizophrenia. Impairments in odor detection and odor identification are present early in the course of illness and among those at risk for the disorder. These behavioral impairments have been linked to both physiological and anatomical abnormalities in the neural substrates subserving olfaction, including relatively peripheral elements of the olfactory system. The location of olfactory receptor neurons in the nasal epithelium allows noninvasive access to these neurons in living subjects. This offers a unique opportunity to directly assess neuronal integrity in vivo in patients. The peripheral olfactory receptor neuron response to odor stimulation was assessed in 21 schizophrenia patients and 18 healthy comparison subjects. The electroolfactogram, representing the electrical depolarization of the olfactory receptor neurons, was recording following stimulation with different doses and durations of hydrogen sulfide, a pure olfactory nerve stimulant. Schizophrenia patients had abnormally large depolarization responses following odor stimulation, independent of clinical symptomatology, antipsychotic medication dosage or smoking history. Although the precise pathophysiological mechanism is unknown, this olfactory receptor neuron abnormality is consistent with several lines of evidence suggesting altered proliferation or maturation of olfactory receptor neuron cell lineages in schizophrenia. It is also consistent with emerging evidence of disruptions of cyclic AMP-mediated intracellular signaling mechanisms, and may be a marker of these disruptions. It unambiguously demonstrates that neurophysiological disturbances in schizophrenia are not limited to cortical and subcortical structures, but rather include even the most peripheral sensory neurons. PMID:18754006

  5. 5-HT7 Receptors Are Not Involved in Neuropeptide Release in Primary Cultured Rat Trigeminal Ganglion Neurons.

    PubMed

    Wang, Xiaojuan; Hu, Rong; Liang, Jianbo; Li, Ze; Sun, Weiwen; Pan, Xiaoping

    2016-06-01

    Migraine is a common but complex neurological disorder. Its precise mechanisms are not fully understood. Increasing indirect evidence indicates that 5-HT7 receptors may be involved; however, their role remains unknown. Our previous in vivo study showed that selective blockade of 5-HT7 receptors caused decreased serum levels of calcitonin gene-related peptide (CGRP) in the external jugular vein following electrical stimulation of the trigeminal ganglion (TG) in an animal model of migraine. In the present study, we used an in vitro model of cultured TG cells to further investigate whether 5-HT7 receptors are directly responsible for the release of CGRP and substance P from TG neurons. We stimulated rat primary cultured TG neurons with capsaicin or potassium chloride (KCl) to mimic neurogenic inflammation, resulting in release of CGRP and substance P. 5-HT7 receptors were abundantly expressed in TG neurons. Greater than 93 % of 5-HT7 receptor-positive neurons co-expressed CGRP and 56 % co-expressed substance P. Both the capsaicin- and KCl-induced release of CGRP and substance P were unaffected by pretreatment of cultured TG cells with the selective 5-HT7 receptor agonist AS19 and antagonist SB269970. This study demonstrates for the first time that 5-HT7 receptors are abundantly co-expressed with CGRP and substance P in rat primary TG neurons and suggests that they are not responsible for the release of CGRP and substance P from cultured TG neurons evoked by capsaicin or KCl. PMID:26892478

  6. A small cohort of FRUM and Engrailed-expressing neurons mediate successful copulation in Drosophila melanogaster

    PubMed Central

    2013-01-01

    Background In Drosophila, male flies require the expression of the male-specific Fruitless protein (FRUM) within the developing pupal and adult nervous system in order to produce male courtship and copulation behaviors. Recent evidence has shown that specific subsets of FRUM neurons are necessary for particular steps of courtship and copulation. In these neurons, FRUM function has been shown to be important for determining sex-specific neuronal characteristics, such as neurotransmitter profile and morphology. Results We identified a small cohort of FRUM interneurons in the brain and ventral nerve cord by their co-expression with the transcription factor Engrailed (En). We used an En-GAL4 driver to express a fruM RNAi construct in order to selectively deplete FRUM in these En/FRUM co-expressing neurons. In courtship and copulation tests, these males performed male courtship at wild-type levels but were frequently sterile. Sterility was a behavioral phenotype as these En-fruMRNAi males were less able to convert a copulation attempt into a stable copulation, or did not maintain copulation for long enough to transfer sperm and/or seminal fluid. Conclusions We have identified a population of interneurons necessary for successful copulation in Drosophila. These data confirm a model in which subsets of FRUM neurons participate in independent neuronal circuits necessary for individual steps of male behavior. In addition, we have determined that these neurons in wild-type males have homologues in females and fru mutants, with similar placement, projection patterns, and neurochemical profiles. PMID:23688386

  7. A Genome-Wide Association Study for Culm Cellulose Content in Barley Reveals Candidate Genes Co-Expressed with Members of the CELLULOSE SYNTHASE A Gene Family

    PubMed Central

    Houston, Kelly; Burton, Rachel A.; Sznajder, Beata; Rafalski, Antoni J.; Dhugga, Kanwarpal S.; Mather, Diane E.; Taylor, Jillian; Steffenson, Brian J.; Waugh, Robbie; Fincher, Geoffrey B.

    2015-01-01

    Cellulose is a fundamentally important component of cell walls of higher plants. It provides a scaffold that allows the development and growth of the plant to occur in an ordered fashion. Cellulose also provides mechanical strength, which is crucial for both normal development and to enable the plant to withstand both abiotic and biotic stresses. We quantified the cellulose concentration in the culm of 288 two – rowed and 288 six – rowed spring type barley accessions that were part of the USDA funded barley Coordinated Agricultural Project (CAP) program in the USA. When the population structure of these accessions was analysed we identified six distinct populations, four of which we considered to be comprised of a sufficient number of accessions to be suitable for genome-wide association studies (GWAS). These lines had been genotyped with 3072 SNPs so we combined the trait and genetic data to carry out GWAS. The analysis allowed us to identify regions of the genome containing significant associations between molecular markers and cellulose concentration data, including one region cross-validated in multiple populations. To identify candidate genes we assembled the gene content of these regions and used these to query a comprehensive RNA-seq based gene expression atlas. This provided us with gene annotations and associated expression data across multiple tissues, which allowed us to formulate a supported list of candidate genes that regulate cellulose biosynthesis. Several regions identified by our analysis contain genes that are co-expressed with CELLULOSE SYNTHASE A (HvCesA) across a range of tissues and developmental stages. These genes are involved in both primary and secondary cell wall development. In addition, genes that have been previously linked with cellulose synthesis by biochemical methods, such as HvCOBRA, a gene of unknown function, were also associated with cellulose levels in the association panel. Our analyses provide new insights into the

  8. Postnatal development of nestin positive neurons in rat basal forebrain: different onset and topography with choline acetyltransferase and parvalbumin expression.

    PubMed

    Guo, Kai-Hua; Li, Dong-Pei; Gu, Huai-Yu; Jie-Xu; Yao, Zhi-Bin

    2014-06-01

    Our previous studies identified a sub-population of cholinergic neurons which express nestin in the rostral part of the basal forebrain (BF) in normal adult rats. In the present study, the postnatal developmental patterns of nestin, choline acetyl transferase (ChAT) and parvalbumin (PV) positive neurons were explored by means of immunohistochemistry combined with immunofluorescence double label methods. Compared with early onset of ChAT expression (from P1) and delayed onset of PV expression (from P16), nestin positive activity was detected in the BF from P9 and co-expressed by parts of the ChAT positive neurons within the same region during the whole postnatal development process. However, ChAT and PV were not coexpressed by the neurons within the medial septum-diagonal band of Broca (MS-DBB) of BF. These results might imply a composite of separate development patterns displayed by different subpopulations of cholinergic neurons (nestin positive cholinergic neurons and nestin negative cholinergic neurons) within this region. Moreover, the topographic distribution of nestin, ChAT and PV positive neurons also showed different characteristics. In summary, our present study revealed a remarkable timing and topographic difference on the postnatal development of the nestin expression within the MS-DBB of BF compared with ChAT and PV expression. It is further suggested that nestin is re-expressed by cholinergic neurons in the BF after differentiation but not persisted from neuronal precursor cells. PMID:24657285

  9. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

    PubMed Central

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  10. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons.

    PubMed

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  11. Gammaherpesvirus Infection of Human Neuronal Cells

    PubMed Central

    Jha, Hem Chandra; Mehta, Devan; Lu, Jie; El-Naccache, Darine; Shukla, Sanket K.; Kovacsics, Colleen; Kolson, Dennis

    2015-01-01

    ABSTRACT Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. PMID:26628726

  12. Neuroanatomical Evidence That Kisspeptin Directly Regulates Isotocin and Vasotocin Neurons

    PubMed Central

    Kanda, Shinji; Akazome, Yasuhisa; Mitani, Yuta; Okubo, Kataaki; Oka, Yoshitaka

    2013-01-01

    Neuropeptide kisspeptin has been suggested to be an essential central regulator of reproduction in response to changes in serum gonadal steroid concentrations. However, in spite of wide kisspeptin receptor distribution in the brain, especially in the preoptic area and hypothalamus, the research focus has mostly been confined to the kisspeptin regulation on GnRH neurons. Here, by using medaka whose kisspeptin (kiss1) neurons have been clearly demonstrated to be regulated by sex steroids, we analyzed the anatomical distribution of kisspeptin receptors Gpr54-1 and Gpr54-2. Because the both receptors were shown to be activated by kisspeptins (Kiss1 and Kiss2), we analyzed the anatomical distribution of the both receptors by in situ hybridization. They were mainly expressed in the ventral telencephalon, preoptic area, and hypothalamus, which have been suggested to be involved in homeostatic functions including reproduction. First, we found gpr54-2 mRNA expression in nucleus preopticus pars magnocellularis and demonstrated that vasotocin and isotocin (Vasopressin and Oxytocin ortholog, respectively) neurons express gpr54-2 by dual in situ hybridization. Given that kisspeptin administration increases serum oxytocin and vasopressin concentration in mammals, the present finding are likely to be vertebrate-wide phenomenon, although direct regulation has not yet been demonstrated in mammals. We then analyzed co-expression of kisspeptin receptors in three types of GnRH neurons. It was clearly demonstrated that gpr54-expressing cells were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves. In contrast, there was no gpr54-expressing cell in the vicinities of neuromodulatory GnRH2 or GnRH3 neurons. From these results, we suggest that medaka kisspeptin neurons directly regulate some behavioral and neuroendocrine functions via vasotocin/isotocin neurons, whereas they do not regulate hypophysiotropic GnRH1 neurons at least in a direct manner. Thus

  13. Calretinin as a Marker for Premotor Neurons Involved in Upgaze in Human Brainstem

    PubMed Central

    Adamczyk, Christopher; Strupp, Michael; Jahn, Klaus; Horn, Anja K. E.

    2015-01-01

    Eye movements are generated by different premotor pathways. Damage to them can cause specific deficits of eye movements, such as saccades. For correlative clinico-anatomical post-mortem studies of cases with eye movement disorders it is essential to identify the functional cell groups of the oculomotor system in the human brain by marker proteins. Based on monkey studies, the premotor neurons of the saccadic system can be identified by the histochemical markers parvalbumin (PAV) and perineuronal nets in humans. These areas involve the interstitial nucleus of Cajal (INC) and the rostral interstitial nucleus of the medial longitudinal fascicle (RIMLF), which both contain premotor neurons for upgaze and downgaze. Recent monkey and human studies revealed a selective excitatory calretinin (CR)-positive input to the motoneurons mediating upgaze, but not to those for downgaze. Three premotor regions were identified as sources of CR input in monkey: y-group, INC and RIMLF. These findings suggest that the expression pattern of parvalbumin and CR may help to identify premotor neurons involved in up- or downgaze. In a post-mortem study of five human cases without neurological diseases we investigated the y-group, INC and RIMLF for the presence of parvalbumin and CR positive neurons including their co-expression. Adjacent thin paraffin sections were stained for the aggrecan (ACAN) component of perineuronal nets, parvalbumin or CR and glutamate decarboxylase. The comparative analysis of scanned thin sections of INC and RIMLF revealed medium-sized parvalbumin positive neurons with and without CR coexpression, which were intermingled. The parvalbumin/CR positive neurons in both nuclei are considered as excitatory premotor upgaze neurons. Accordingly, the parvalbumin-positive neurons lacking CR are considered as premotor downgaze neurons in RIMLF, but may in addition include inhibitory premotor upgaze neurons in the INC as indicated by co-expression of glutamate decarboxylase in a

  14. Systems toxicology of chemically induced liver and kidney injuries: histopathology-associated gene co-expression modules.

    PubMed

    Te, Jerez A; AbdulHameed, Mohamed Diwan M; Wallqvist, Anders

    2016-09-01

    Organ injuries caused by environmental chemical exposures or use of pharmaceutical drugs pose a serious health risk that may be difficult to assess because of a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific histopathology outcomes via biomarkers will provide a foundation for designing precise and robust diagnostic tests. We identified co-expressed genes (modules) specific to injury endpoints using the Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) - a toxicogenomics database containing organ-specific gene expression data matched to dose- and time-dependent chemical exposures and adverse histopathology assessments in Sprague-Dawley rats. We proposed a protocol for selecting gene modules associated with chemical-induced injuries that classify 11 liver and eight kidney histopathology endpoints based on dose-dependent activation of the identified modules. We showed that the activation of the modules for a particular chemical exposure condition, i.e., chemical-time-dose combination, correlated with the severity of histopathological damage in a dose-dependent manner. Furthermore, the modules could distinguish different types of injuries caused by chemical exposures as well as determine whether the injury module activation was specific to the tissue of origin (liver and kidney). The generated modules provide a link between toxic chemical exposures, different molecular initiating events among underlying molecular pathways and resultant organ damage. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons, Ltd. PMID:26725466

  15. Gamma-Retroviral Vector Design for the Co-Expression of Artificial microRNAs and Therapeutic Proteins

    PubMed Central

    Park, Tristen S.; Zhang, Ling; Zheng, Zhili; Morgan, Richard A.

    2014-01-01

    To generate γ-retroviral vectors for stable conjoint expression of artificial microRNAs (amiR) and therapeutic genes in primary human lymphocytes, and to identify the design parameters that are key for successful vector generation. Gamma-retroviral vectors were designed to co-express both amiRs and a linked reporter gene, truncated CD34 (tCD34). Artificial miRs based on microRNAs miR-16, miR-142, miR-146b, miR-150, miR155, and miR-223 were inserted into sites within the intron of the vector and tested for tCD34 expression by flow cytometry (FACS). Different constructs were assembled with amiRs targeted to knockdown expression of suppressor of cytokine signaling 1 (SOCS1) or programmed cell death 1 (PDCD1, PD-1). Three of the six amiRs maintained tCD34 expression. Expansion of primary human T cells transduced with these amiR vectors, as well as transgene expression, were equivalent to control engineered T cells over a 40-day period. Knockdown of SOCS1 RNA and PD-1 expression by FACS was shown to vary between constructs, dependent on either the specific short interfering RNA sequence used in the amiR, or the microRNA backbone and location in the vector intron. Gamma-retroviral vectors that both efficiently knockdown endogenous gene expression and maintain linked transgene production can be produced, but empirical vector evaluations were best suited for optimal construct analysis. PMID:25019196

  16. Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli.

    PubMed

    Jiang, Min; Chen, Xu; Liang, Liya; Liu, Rongming; Wan, Qing; Wu, Mingke; Zhang, Hanwen; Ma, Jiangfeng; Chen, Kequan; Ouyang, Pingkai

    2014-03-01

    Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars. PMID:24564896

  17. Mining Temporal Protein Complex Based on the Dynamic PIN Weighted with Connected Affinity and Gene Co-Expression

    PubMed Central

    Shen, Xianjun; Jiang, Xingpeng; He, Tingting; Hu, Xiaohua; Yang, Jincai

    2016-01-01

    The identification of temporal protein complexes would make great contribution to our knowledge of the dynamic organization characteristics in protein interaction networks (PINs). Recent studies have focused on integrating gene expression data into static PIN to construct dynamic PIN which reveals the dynamic evolutionary procedure of protein interactions, but they fail in practice for recognizing the active time points of proteins with low or high expression levels. We construct a Time-Evolving PIN (TEPIN) with a novel method called Deviation Degree, which is designed to identify the active time points of proteins based on the deviation degree of their own expression values. Owing to the differences between protein interactions, moreover, we weight TEPIN with connected affinity and gene co-expression to quantify the degree of these interactions. To validate the efficiencies of our methods, ClusterONE, CAMSE and MCL algorithms are applied on the TEPIN, DPIN (a dynamic PIN constructed with state-of-the-art three-sigma method) and SPIN (the original static PIN) to detect temporal protein complexes. Each algorithm on our TEPIN outperforms that on other networks in terms of match degree, sensitivity, specificity, F-measure and function enrichment etc. In conclusion, our Deviation Degree method successfully eliminates the disadvantages which exist in the previous state-of-the-art dynamic PIN construction methods. Moreover, the biological nature of protein interactions can be well described in our weighted network. Weighted TEPIN is a useful approach for detecting temporal protein complexes and revealing the dynamic protein assembly process for cellular organization. PMID:27100396

  18. The evaluative value of Sema3C and MFN2 co-expression detected by immunohistochemistry for prognosis in hepatocellular carcinoma patients after hepatectomy

    PubMed Central

    Feng, Xu; Zhu, Kelei; Liu, Jinghua; Chen, Jiang; Tang, Jiacheng; Liang, Yuelong; Jin, Renan; Liang, Xiao; Cai, Xiujun

    2016-01-01

    Background The ability to evaluate the prognosis of hepatocellular carcinoma (HCC) following hepatectomy using biological markers is of great importance. Materials and methods In this study, we collected samples from 54 patients with HCC after hepatectomy. Immunohistochemistry was used to detect the expression of Sema3C and MFN2 in the HCC samples. Results Immunohistochemistry results showed that Sema3C and MFN2 co-expression was significantly associated with tumor size. In addition, a significant association between high Sema3C and low MFN2 levels and shorter overall survival was noted, when Sema3C and MFN2 co-expression was analyzed. Conclusion The results suggest that the correlative expression level of Sema3C and MFN2 has a strong value in the prognosis of patients with HCC following hepatectomy. PMID:27313467

  19. Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage*

    PubMed Central

    Lim, Mi-Sun; Chang, Mi-Yoon; Kim, Sang-Mi; Yi, Sang-Hoon; Suh-Kim, Haeyoung; Jung, Sung Jun; Kim, Min Jung; Kim, Jin Hyuk; Lee, Yong-Sung; Lee, Soo Young; Kim, Dong-Wook; Lee, Sang-Hun; Park, Chang-Hwan

    2015-01-01

    Recent groundbreaking work has demonstrated that combined expression of the transcription factors Brn2, Ascl1, and Myt1L (BAM; also known as Wernig factors) convert mouse fibroblasts into postmitotic neuronal cells. However, questions remain regarding whether trans-conversion is achieved directly or involves an intermediary precursor stage. Trans-conversion toward expandable neural precursor cells (NPCs) is more useful than direct one-step neuron formation with respect to yielding a sufficient number of cells and the feasibility of manipulating NPC differentiation toward certain neuron subtypes. Here, we show that co-expression of Wernig factors and Bcl-xL induces fibroblast conversion into NPCs (induced NPCs (iNPCs)) that are highly expandable for >100 passages. Gene expression analyses showed that the iNPCs exhibited high expression of common NPC genes but not genes specific to defined embryonic brain regions. This finding indicated that a regional identity of iNPCs was not established. Upon induction, iNPCs predominantly differentiated into astrocytes. However, the differentiation potential was not fixed and could be efficiently manipulated into general or specific subtypes of neurons by expression of additional genes. Specifically, overexpression of Nurr1 and Foxa2, transcription factors specific for midbrain dopamine neuron development, drove iNPCs to yield mature midbrain dopamine neurons equipped with presynaptic DA neuronal functions. We further assessed the therapeutic potential of iNPCs in Parkinson disease model rats. PMID:26023233

  20. Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

    PubMed

    Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun

    2016-01-01

    Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. PMID:25865630

  1. Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development

    PubMed Central

    Zhao, Yunxia; Hou, Ye; Zhao, Changzhi; Liu, Fei; Luan, Yu; Jing, Lu; Li, Xinyun; Zhu, Mengjin; Zhao, Shuhong

    2016-01-01

    Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs. PMID:27489504

  2. Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development.

    PubMed

    Zhao, Yunxia; Hou, Ye; Zhao, Changzhi; Liu, Fei; Luan, Yu; Jing, Lu; Li, Xinyun; Zhu, Mengjin; Zhao, Shuhong

    2016-01-01

    Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs. PMID:27489504

  3. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.)

    PubMed Central

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet’s salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na+/H+ antiporter and H+-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na+ and K+ in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na+-toxicity for plants. PMID:26284097

  4. Efficient multicistronic co-expression of hNIS and hTPO in prostate cancer cells for nonthyroidal tumor radioiodine therapy.

    PubMed

    Li, Guoquan; Xiang, Lei; Yang, Weidong; Wang, Zhe; Wang, Jing; Chen, Kai

    2012-01-01

    Radioiodine therapy has proven to be a safe and effective approach in the treatment of differentiated thyroid cancer. Similar treatment strategies have been exploited in nonthyroidal malignancies by transfecting hNIS gene into tumor cells or xenografts. However, rapid radioiodine efflux is often observed after radioiodine uptake, limiting the overall antitumor effects. In this study, we aimed at constructing multicistronic co-expression of hNIS and hTPO genes in tumor cells to enhance the radioiodine uptake and prolong the radioiodine retention. Driven by the cytomegalovirus promoter, hNIS and hTPO were simultaneously inserted into the expression cassette of adenoviral vector. An Ad5 viral vector (Ad-CMV-hTPO-T2A-hNIS) was assembled as a gene therapy vehicle by Gateway technology and 2A method. The co-expression of hNIS and hTPO genes was confirmed by a double-label immunofluorescence assay. The radioiodine ((125)I) uptake and efflux effects induced by co-expression of hNIS and hTPO genes were determined in transfected and non-transfected PC-3 cells. Significantly higher uptake (6.58 ± 0.56 fold, at 1 h post-incubation) and prolonged retention (5.47 ± 0.36 fold, at 1 h of cell efflux) of radioiodine ((125)I) were observed in hNIS and hTPO co-expressed PC-3 cells as compared to non-transfected PC-3 cells. We concluded that the new virus vector displayed favorable radioiodine uptake and retention properties in hNIS-hTPO transfected PC-3 cells. Our study will provide valuable information on improving the efficacy of hNIS-hTPO co-mediated radioiodine gene therapy. PMID:23145364

  5. Clinical significance of co-expression of MYC and BCL2 protein in aggressive B-cell lymphomas treated with a second line immunochemotherapy.

    PubMed

    Miura, Katsuhiro; Takahashi, Hiromichi; Nakagawa, Masaru; Izu, Asami; Sugitani, Masahiko; Kurita, Daisuke; Sakagami, Masashi; Ohtake, Shimon; Uchino, Yoshihito; Hojo, Atsuko; Kodaira, Hitomi; Yagi, Mai; Kobayashi, Yujin; Iriyama, Noriyoshi; Kobayashi, Sumiko; Kiso, Satomi; Hirabayashi, Yukio; Hatta, Yoshihiro; Takei, Masami

    2016-06-01

    The clinical significance of concurrent expression of MYC and BCL2 protein, known as "double-expressor lymphoma" (DEL), among patients with relapsed or refractory aggressive B-cell lymphomas, remains unclear. A retrospective analysis was performed of 38 patients treated with a salvage treatment consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone followed by consolidative high-dose chemotherapies. A total of 17 cases (45%) were categorized as DEL using immunohistochemical assay with a cut-off value of positivity of 40% for MYC and 50% for BCL2, respectively. DEL was associated with a lower overall response rate (35% vs 71%, p = 0.0481), worse 2-year progression-free survival (9% vs 67%, p = 0.001) and overall survival (35% vs 71%, p = 0.037). This analysis suggests that DEL is common among patients with relapsed/refractory aggressive B-cell lymphomas and that such patients require novel treatment strategies. PMID:26390147

  6. The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co-expressed during B cell development.

    PubMed

    Dul, J L; Argon, Y; Winkler, T; ten Boekel, E; Melchers, F; Mårtensson, I L

    1996-04-01

    The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16-17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre

  7. Channel Properties of Nax Expressed in Neurons

    PubMed Central

    Matsumoto, Masahito; Hiyama, Takeshi Y.; Kuboyama, Kazuya; Suzuki, Ryoko; Fujikawa, Akihiro; Noda, Masaharu

    2015-01-01

    Nax is a sodium-concentration ([Na+])-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o) in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95) through its PSD95/Disc-large/ZO-1 (PDZ)-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells. PMID:25961826

  8. Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli.

    PubMed

    Peng, Shuaiying; Chu, Zhongmei; Lu, Jianfeng; Li, Dongxiao; Wang, Yonghong; Yang, Shengli; Zhang, Yi

    2016-05-01

    The extracellular α-amylase from the hyperthermophilic archaeum Pyrococcus furiosus (PFA) is extremely thermostable and of an industrial importance and interest. PFA aggregates and accumulates as insoluble inclusion bodies when expressed as a heterologous protein at a high level in Escherichia coli. In the present study, we investigated the roles of chaperones from P. furiosus in the soluble expression of recombinant PFA in E. coli. The results indicate that co-expression of PFA with the molecular chaperone prefoldin alone significantly increased the soluble expression of PFA. Although, co-expression of other main chaperone components from P. furiosus, such as the small heat shock protein (sHSP) or chaperonin (HSP60), was also able to improve the soluble expression of PFA to a certain extent. Co-expression of chaperonin or sHSP in addition to prefoldin did not further increase the soluble expression of PFA. This finding emphasizes the biotechnological potentials of the molecular chaperone prefoldin from P. furiosus, which may facilitate the production of recombinant PFA. PMID:26862080

  9. Significant enhancement of methionol production by co-expression of the aminotransferase gene ARO8 and the decarboxylase gene ARO10 in Saccharomyces cerevisiae.

    PubMed

    Yin, Sheng; Lang, Tiandan; Xiao, Xiao; Liu, Li; Sun, Baoguo; Wang, Chengtao

    2015-03-01

    Methionol is an important volatile sulfur flavor compound, which can be produced via the Ehrlich pathway in Saccharomyces cerevisiae. Aminotransferase and decarboxylase are essential enzymes catalyzing methionol biosynthesis. In this work, two aminotransferase genes ARO8 and ARO9 and one decarboxylase gene ARO10 were introduced into S. cerevisiae S288c, respectively, via an expression vector. Over-expression of ARO8 resulted in higher aminotransferase activity than that of ARO9. And the cellular decarboxylase activity was remarkably increased by over-expression of ARO10. A co-expression vector carrying both ARO8 and ARO10 was further constructed to generate the recombinant strain S810. Shaking flask experiments showed that the methionol yield from S810 reached 1.27 g L(-1), which was increased by 51.8 and 68.8% compared to that from the wild-type strain and the control strain harboring the empty vector. The fed-batch fermentation by strain S810 produced 3.24 g L(-1) of methionol after 72 h of cultivation in a bioreactor. These results demonstrated that co-expression of ARO8 and ARO10 significantly boosted the methionol production. It is the first time that more than 3.0 g L(-1) of methionol produced by genetically engineered yeast strain was reported by co-expression of the aminotransferase and decarboxylase via the Ehrlich pathway. PMID:25743068

  10. Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

    PubMed

    Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-10-01

    D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. PMID:25152409

  11. Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

    PubMed Central

    García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

    2015-01-01

    Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

  12. Co-expression of ING4 and P53 enhances hypopharyngeal cancer chemosensitivity to cisplatin in vivo

    PubMed Central

    Ren, Xin; Liu, Hao; Zhang, Mingjie; Wang, Mengjun; Ma, Shiyin

    2016-01-01

    Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co-expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad-ING4-P53, cisplatin, or a combination of Ad-ING4-P53 and cisplatin (Ad-ING4-P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription-polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad-ING4-P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad-ING4-P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl-2 was decreased in the Ad-ING4-P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy

  13. High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli.

    PubMed

    Mahamad, Pornpimol; Boonchird, Chuenchit; Panbangred, Watanalai

    2016-07-01

    CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 ± 8.95 μg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 μg/mL and 150.07 ± 8.13 μg/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host. PMID:27020286

  14. Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function.

    PubMed

    Zuure, Wieteke A; Roberts, Amy L; Quennell, Janette H; Anderson, Greg M

    2013-11-01

    The adipocyte-derived hormone leptin acts in the brain to modulate the central driver of fertility: the gonadotropin releasing hormone (GnRH) neuronal system. This effect is indirect, as GnRH neurons do not express leptin receptors (LEPRs). Here we test whether GABAergic or glutamatergic neurons provide the intermediate pathway between the site of leptin action and the GnRH neurons. Leptin receptors were deleted from GABA and glutamate neurons using Cre-Lox transgenics, and the downstream effects on puberty onset and reproduction were examined. Both mouse lines displayed the expected increase in body weight and region-specific loss of leptin signaling in the hypothalamus. The GABA neuron-specific LEPR knock-out females and males showed significantly delayed puberty onset. Adult fertility observations revealed that these knock-out animals have decreased fecundity. In contrast, glutamate neuron-specific LEPR knock-out mice displayed normal fertility. Assessment of the estrogenic hypothalamic-pituitary-gonadal axis regulation in females showed that leptin action on GABA neurons is not necessary for estradiol-mediated suppression of tonic luteinizing hormone secretion (an indirect measure of GnRH neuron activity) but is required for regulation of a full preovulatory-like luteinizing hormone surge. In conclusion, leptin signaling in GABAergic (but not glutamatergic neurons) plays a critical role in the timing of puberty onset and is involved in fertility regulation throughout adulthood in both sexes. These results form an important step in explaining the role of central leptin signaling in the reproductive system. Limiting the leptin-to-GnRH mediators to GABAergic cells will enable future research to focus on a few specific types of neurons. PMID:24198376

  15. The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping.

    PubMed

    Braegger, Fiona E; Asarian, Lori; Dahl, Kirsten; Lutz, Thomas A; Boyle, Christina N

    2014-10-01

    Amylin reduces meal size by activating noradrenergic neurons in the area postrema (AP). Neurons in the AP also mediate the eating-inhibitory effects of salmon calcitonin (sCT), a potent amylin agonist, but the phenotypes of the neurons mediating its effect are unknown. Here we investigated whether sCT activates similar neuronal populations to amylin, and if its anorectic properties also depend on AP function. Male rats underwent AP lesion (APX) or sham surgery. Meal patterns were analysed under ad libitum and post-deprivation conditions. The importance of the AP in mediating the anorectic action of sCT was examined in feeding experiments of dose-response effects of sCT in APX vs. sham rats. The effect of sCT to induce Fos expression was compared between surgery groups, and relative to amylin. The phenotype of Fos-expressing neurons in the brainstem was examined by testing for the co-expression of dopamine beta hydroxylase (DBH) or tryptophan hydroxylase (TPH). By measuring the apposition of vesicular glutamate transporter-2 (VGLUT2)-positive boutons, potential glutamatergic input to amylin- and sCT-activated AP neurons was compared. Similar to amylin, an intact AP was necessary for sCT to reduce eating. Further, co-expression between Fos activation and DBH after amylin or sCT did not differ markedly, while co-localization of Fos and TPH was minor. Approximately 95% of neurons expressing Fos and DBH after amylin or sCT treatment were closely apposed to VGLUT2-positive boutons. Our study suggests that the hindbrain pathways engaged by amylin and sCT share many similarities, including the mediation by AP neurons. PMID:25040689

  16. Tinbergen on mirror neurons

    PubMed Central

    Heyes, Cecilia

    2014-01-01

    Fifty years ago, Niko Tinbergen defined the scope of behavioural biology with his four problems: causation, ontogeny, survival value and evolution. About 20 years ago, there was another highly significant development in behavioural biology—the discovery of mirror neurons (MNs). Here, I use Tinbergen's original four problems (rather than the list that appears in textbooks) to highlight the differences between two prominent accounts of MNs, the genetic and associative accounts; to suggest that the latter provides the defeasible ‘best explanation’ for current data on the causation and ontogeny of MNs; and to argue that functional analysis, of the kind that Tinbergen identified somewhat misleadingly with studies of ‘survival value’, should be a high priority for future research. In this kind of functional analysis, system-level theories would assign MNs a small, but potentially important, role in the achievement of action understanding—or another social cognitive function—by a production line of interacting component processes. These theories would be tested by experimental intervention in human and non-human animal samples with carefully documented and controlled developmental histories. PMID:24778376

  17. Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

    PubMed Central

    Bosch, Marie K.; Nerbonne, Jeanne M.; Ornitz, David M.

    2014-01-01

    Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo. PMID:25093726

  18. Mesmerising mirror neurons.

    PubMed

    Heyes, Cecilia

    2010-06-01

    Mirror neurons have been hailed as the key to understanding social cognition. I argue that three currents of thought-relating to evolution, atomism and telepathy-have magnified the perceived importance of mirror neurons. When they are understood to be a product of associative learning, rather than an adaptation for social cognition, mirror neurons are no longer mesmerising, but they continue to raise important questions about both the psychology of science and the neural bases of social cognition. PMID:20167276

  19. Evolution of neoplastic development in the liver of transgenic mice co-expressing c-myc and transforming growth factor-alpha.

    PubMed Central

    Santoni-Rugiu, E.; Nagy, P.; Jensen, M. R.; Factor, V. M.; Thorgeirsson, S. S.

    1996-01-01

    We have previously shown that co-expression of c-myc and transforming growth factor (TGF)-alpha as transgenes in mouse liver results in major enhancement of neoplastic development in this organ as compared with expression of either of these transgenes alone. In this report we describe in detail the progression from liver cell dysplasia to hepatocellular carcinomas (HCCs) occurring in the liver of c-myc/TGF-alpha and c-myc transgenic mice. Despite morphological similarities in the sequence of events between the two transgenic lines, the dramatic acceleration, extent, and severity of hepatic lesions in c-myc/TGF-alpha mice clearly demonstrated the synergistic effects of this transgenic combination. Although c-myc/TGF-alpha and c-myc females displayed longer latency and lower tumor incidence, the pathological changes were the same as those seen in the male mice, including the formation of HCCs, which are absent in TGF-alpha single-transgenic females. Tumors in single- and double-transgenic mice showed induction of the endogenous c-myc and TGF-alpha and, most frequently, unchanged or decreased epidermal growth factor receptor, further indicating the collaborative role of c-myc and TGF-alpha in providing a selective growth advantage to tumor cells independently of the epidermal growth factor receptor levels. To identify possible tumor precursors, we focused particularly on the dysplastic changes preceding and accompanying the appearance of preneoplastic and neoplastic lesions in the double-transgenic mice. Early on, these changes were characterized by the appearance of large dysplastic hepatocytes, mostly pericentrally, expressing high levels of TGF-alpha and uPA, as well as TGF-beta 1, particularly in apoptotic cells. After a short period of replication and expansion into the liver parenchyma, as well as penetration into the central veins, these cells underwent apoptotic cell death while preneoplastic and neoplastic lesions were forming. The peritumorous tissues also

  20. Patterning human neuronal networks on photolithographically engineered silicon dioxide substrates functionalized with glial analogues.

    PubMed

    Hughes, Mark A; Brennan, Paul M; Bunting, Andrew S; Cameron, Katherine; Murray, Alan F; Shipston, Mike J

    2014-05-01

    Interfacing neurons with silicon semiconductors is a challenge being tackled through various bioengineering approaches. Such constructs inform our understanding of neuronal coding and learning and ultimately guide us toward creating intelligent neuroprostheses. A fundamental prerequisite is to dictate the spatial organization of neuronal cells. We sought to pattern neurons using photolithographically defined arrays of polymer parylene-C, activated with fetal calf serum. We used a purified human neuronal cell line [Lund human mesencephalic (LUHMES)] to establish whether neurons remain viable when isolated on-chip or whether they require a supporting cell substrate. When cultured in isolation, LUHMES neurons failed to pattern and did not show any morphological signs of differentiation. We therefore sought a cell type with which to prepattern parylene regions, hypothesizing that this cellular template would enable secondary neuronal adhesion and network formation. From a range of cell lines tested, human embryonal kidney (HEK) 293 cells patterned with highest accuracy. LUHMES neurons adhered to pre-established HEK 293 cell clusters and this coculture environment promoted morphological differentiation of neurons. Neurites extended between islands of adherent cell somata, creating an orthogonally arranged neuronal network. HEK 293 cells appear to fulfill a role analogous to glia, dictating cell adhesion, and generating an environment conducive to neuronal survival. We next replaced HEK 293 cells with slower growing glioma-derived precursors. These primary human cells patterned accurately on parylene and provided a similarly effective scaffold for neuronal adhesion. These findings advance the use of this microfabrication-compatible platform for neuronal patterning. PMID:23733444

  1. Characterization of dendritic morphology and neurotransmitter phenotype of thoracic descending propriospinal neurons after complete spinal cord transection and GDNF treatment.

    PubMed

    Deng, Lingxiao; Ruan, Yiwen; Chen, Chen; Frye, Christian Corbin; Xiong, Wenhui; Jin, Xiaoming; Jones, Kathryn; Sengelaub, Dale; Xu, Xiao-Ming

    2016-03-01

    After spinal cord injury (SCI), poor regeneration of damaged axons of the central nervous system (CNS) causes limited functional recovery. This limited spontaneous functional recovery has been attributed, to a large extent, to the plasticity of propriospinal neurons, especially the descending propriospinal neurons (dPSNs). Compared with the supraspinal counterparts, dPSNs have displayed significantly greater regenerative capacity, which can be further enhanced by glial cell line-derived neurotrophic factor (GDNF). In the present study, we applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of dPSNs. We also investigated the neurotransmitters expressed by dPSNs after labeling with a retrograde tracer Fluoro-Gold (FG). dPSNs were examined in animals with sham injuries or complete spinal transections with or without GDNF treatment. Bilateral injections of G-Rabies and FG were made into the 2nd lumbar (L2) spinal cord at 3 days prior to a spinal cord transection performed at the 11th thoracic level (T11). The lesion gap was filled with Gelfoam containing either saline or GDNF in the injury groups. Four days post-injury, the rats were sacrificed for analysis. For those animals receiving G-rabies injection, the GFP signal in the T7-9 spinal cord was visualized via 2-photon microscopy. Dendritic morphology from stack images was traced and analyzed using a Neurolucida software. We found that dPSNs in sham injured animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution with dorsal-ventral retraction and lateral-medial extension. Treatment with GDNF significantly increased the terminal dendritic length of dPSNs. The density of spine-like structures was increased after injury, and treatment with GDNF enhanced this effect. For the group receiving FG injections, immunohistochemistry for glutamate, choline

  2. Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

    PubMed Central

    Kruse, Martin; Vivas, Oscar; Traynor-Kaplan, Alexis

    2016-01-01

    In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons. PMID:26818524

  3. Sensory neuron-specific GPCRs Mrgprs are itch receptors mediating chloroquine-induced pruritus

    PubMed Central

    Liu, Qin; Tang, Zongxiang; Surdenikova, Lenka; Kim, Seungil; Patel, Kush N.; Kim, Andrew; Ru, Fei; Guan, Yun; Weng, Hao-Jui; Geng, Yixun; Undem, Bradley J.; Kollarik, Marian; Chen, Zhou-Feng; Anderson, David J.; Dong, Xinzhong

    2010-01-01

    SUMMARY The cellular and molecular mechanisms mediating histamine-independent itch in primary sensory neurons are largely unknown. Itch induced by chloroquine (CQ) is a common side-effect of this widely used anti-malarial drug. Here we show that Mrgprs, a family of G protein-coupled receptors expressed exclusively in peripheral sensory neurons, function as itch receptors. Mice lacking a cluster of Mrgpr genes display significant deficits in itch induced by CQ but not histamine. CQ directly excites sensory neurons in an Mrgpr-dependent manner. CQ specifically activates mouse MrgprA3 and human MrgprX1. Loss- and gain-of-function studies demonstrate that MrgprA3 is required for CQ responsiveness in mice. Furthermore, MrgprA3-expressing neurons respond to histamine and co-express Gastrin-Releasing Peptide, a peptide involved in itch sensation, and MrgprC11. Activation of these neurons with MrgprC11-specific agonist BAM8-22 induces itch in wild-type but not mutant mice. Therefore, Mrgprs may provide molecular access to itch-selective neurons and constitute novel targets for itch therapeutics. PMID:20004959

  4. RBFOX1 regulates both splicing and transcriptional networks in human neuronal development

    PubMed Central

    Fogel, Brent L.; Wexler, Eric; Wahnich, Amanda; Friedrich, Tara; Vijayendran, Chandran; Gao, Fuying; Parikshak, Neelroop; Konopka, Genevieve; Geschwind, Daniel H.

    2012-01-01

    RNA splicing plays a critical role in the programming of neuronal differentiation and, consequently, normal human neurodevelopment, and its disruption may underlie neurodevelopmental and neuropsychiatric disorders. The RNA-binding protein, fox-1 homolog (RBFOX1; also termed A2BP1 or FOX1), is a neuron-specific splicing factor predicted to regulate neuronal splicing networks clinically implicated in neurodevelopmental disease, including autism spectrum disorder (ASD), but only a few targets have been experimentally identified. We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins. Downstream alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. Several of these differentially expressed genes are further implicated in ASD and related neurodevelopmental diseases. Weighted gene co-expression network analysis demonstrates a high degree of connectivity among these disease-related genes, highlighting RBFOX1 as a key factor coordinating the regulation of both neurodevelopmentally important alternative splicing events and clinically relevant neuronal transcriptional programs in the development of human neurons. PMID:22730494

  5. Neurokinin-1 receptor antagonism attenuates neuronal activity triggered by stress-induced reinstatement of alcohol seeking.

    PubMed

    Schank, J R; Nelson, B S; Damadzic, R; Tapocik, J D; Yao, M; King, C E; Rowe, K E; Cheng, K; Rice, K C; Heilig, M

    2015-12-01

    Substance P (SP) and its cognate neurokinin-1 receptor (NK1R) are involved in alcohol-related behaviors. We have previously reported that NK1R antagonism attenuates stress-induced reinstatement of alcohol seeking and suppresses escalated alcohol self-administration, but does not affect primary reinforcement or cue-induced reinstatement. Here, we administered an NK1R antagonist or vehicle prior to footshock-induced reinstatement of alcohol seeking, and mapped the resulting neuronal activation using Fos immunohistochemistry. As expected, vehicle treated animals exposed to footshock showed induction of Fos immunoreactivity in several regions of the brain stress circuitry, including the amygdala (AMG), nucleus accumbens (NAC), dorsal raphe nucleus (DR), prefrontal cortex (PFC), and bed nucleus of the stria terminalis (BNST). NK1R antagonism selectively suppressed the stress-induced increase in Fos in the DR and NAC shell. In the DR, Fos-induction by stress largely overlapped with tryptophan hydroxylase (TrpH), indicating activation of serotonergic neurons. Of NAC shell neurons activated during stress-induced reinstatement of alcohol seeking, about 30% co-expressed dynorphin (DYN), while 70% co-expressed enkephalin (ENK). Few (<1%) activated NAC shell neurons coexpressed choline acetyltransferase (ChAT), which labels the cholinergic interneurons of this region. Infusion of the NK1R antagonist L822429 into the NAC shell blocked stress-induced reinstatement of alcohol seeking. In contrast, L822429 infusion into the DR had no effect, suggesting that the influence of NK1R signaling on neuronal activity in the DR is indirect. Taken together, our results outline a potential pathway through which endogenous NK1R activation mediates stress-induced alcohol seeking. PMID:26188146

  6. Mechanisms of Transient Signaling via Short and Long Prolactin Receptor Isoforms in Female and Male Sensory Neurons*

    PubMed Central

    Belugin, Sergei; Diogenes, Anibal R.; Patil, Mayur J.; Ginsburg, Erika; Henry, Michael A.; Akopian, Armen N.

    2013-01-01

    Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells. PMID:24142695

  7. Radial Glial Cell–Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site

    PubMed Central

    Xu, Chundi; Funahashi, Yasuhiro; Watanabe, Takashi; Takano, Tetsuya; Nakamuta, Shinichi; Namba, Takashi

    2015-01-01

    How extracellular cues direct axon–dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)–cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon–dendrite polarization in vivo. Furthermore, the RGC–neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho–Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon–dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia–neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases. SIGNIFICANCE STATEMENT Neurons are highly polarized cell lines typically with a single axon and multiple dendrites, which underlies the ability of integrating and transmitting the information in the brain. How is the axon–dendrite polarity of neurons established in the developing neocortex? Here we show that the N-cadherin-mediated radial glial cell–neuron interaction directs axon–dendrite polarization, the radial glial cell–neuron interaction induces polarized distribution of active RhoA and active Rac1 in neurons, and Rho–Rho-kinase signaling is required for axon–dendrite polarization. Our work advances the overall understanding of how extracellular cues direct axon–dendrite polarization in mouse developing neurons. PMID:26511243

  8. Electrophysiological recordings of patterned rat brain stem slice neurons.

    PubMed

    Lauer, L; Vogt, A; Yeung, C K; Knoll, W; Offenhäusser, A

    2002-08-01

    Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining. PMID:12102183

  9. Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

    PubMed Central

    Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M.; McDonald, Karen A.

    2011-01-01

    Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI. PMID:21954339

  10. Co-Expression of Anti-Rotavirus Proteins (Llama VHH Antibody Fragments) in Lactobacillus: Development and Functionality of Vectors Containing Two Expression Cassettes in Tandem

    PubMed Central

    Günaydın, Gökçe; Álvarez, Beatriz; Lin, Yin; Hammarström, Lennart; Marcotte, Harold

    2014-01-01

    Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3) as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11), by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules. PMID:24781086

  11. Quantum neuron design

    NASA Astrophysics Data System (ADS)

    Behrman, Elizabeth; Steck, James

    2014-03-01

    In previous work, we have developed quantum systems that can learn and do information processing much like artificial neural networks. These learning methods have some advantages over other implementations of quantum computing in that they construct their own algorithms and could be robust to noise and decoherence. Here we take the next step, by designing quantum neurons that have some of the important behaviors of biological neurons, yet have the advantage of being complex valued and having quantum computing power. Our neuron model consists of a two-level system coupled to a Gaussian bath representing the environment. Simulations of a interconnected network of these neurons show that the model can both learn standard AI tasks, as similar networks of classical neurons have been shown to do, and, in addition, perform quantum mechanical calculations.

  12. Functional coupling with cardiac muscle promotes maturation of hPSC-derived sympathetic neurons

    PubMed Central

    Oh, Yohan; Cho, Gun-Sik; Li, Zhe; Hong, Ingie; Zhu, Renjun; Kim, Min-Jeong; Kim, Yong Jun; Tampakakis, Emmanouil; Tung, Leslie; Huganir, Richard; Dong, Xinzhong; Kwon, Chulan; Lee, Gabsang

    2016-01-01

    Summary Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B:eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties, and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX:eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish. PMID:27320040

  13. From SNP co-association to RNA co-expression: Novel insights into gene networks for intramuscular fatty acid composition in porcine

    PubMed Central

    2014-01-01

    Background Fatty acids (FA) play a critical role in energy homeostasis and metabolic diseases; in the context of livestock species, their profile also impacts on meat quality for healthy human consumption. Molecular pathways controlling lipid metabolism are highly interconnected and are not fully understood. Elucidating these molecular processes will aid technological development towards improvement of pork meat quality and increased knowledge of FA metabolism, underpinning metabolic diseases in humans. Results The results from genome-wide association studies (GWAS) across 15 phenotypes were subjected to an Association Weight Matrix (AWM) approach to predict a network of 1,096 genes related to intramuscular FA composition in pigs. To identify the key regulators of FA metabolism, we focused on the minimal set of transcription factors (TF) that the explored the majority of the network topology. Pathway and network analyses pointed towards a trio of TF as key regulators of FA metabolism: NCOA2, FHL2 and EP300. Promoter sequence analyses confirmed that these TF have binding sites for some well-know regulators of lipid and carbohydrate metabolism. For the first time in a non-model species, some of the co-associations observed at the genetic level were validated through co-expression at the transcriptomic level based on real-time PCR of 40 genes in adipose tissue, and a further 55 genes in liver. In particular, liver expression of NCOA2 and EP300 differed between pig breeds (Iberian and Landrace) extreme in terms of fat deposition. Highly clustered co-expression networks in both liver and adipose tissues were observed. EP300 and NCOA2 showed centrality parameters above average in the both networks. Over all genes, co-expression analyses confirmed 28.9% of the AWM predicted gene-gene interactions in liver and 33.0% in adipose tissue. The magnitude of this validation varied across genes, with up to 60.8% of the connections of NCOA2 in adipose tissue being validated via co-expression

  14. Neonatal seizures induced by pentylenetetrazol or kainic acid disrupt primary cilia growth on developing mouse cortical neurons.

    PubMed

    Parker, Alexander K; Le, Megan M; Smith, Tyler S; Hoang-Minh, Lan B; Atkinson, Eric W; Ugartemendia, George; Semple-Rowland, Susan; Coleman, Jason E; Sarkisian, Matthew R

    2016-08-01

    Neonatal or early-life seizures (ELS) are often associated with life-long neurophysiological, cognitive and behavioral deficits, but the underlying mechanisms contributing to these deficits remain poorly understood. Newborn, post-migratory cortical neurons sprout ciliary buds (procilia) that mature into primary cilia. Disruption of the growth or signaling capabilities of these cilia has been linked to atypical neurite outgrowth from neurons and abnormalities in neuronal circuitry. Here, we tested the hypothesis that generalized seizures induced by pentylenetetrazol (PTZ) or kainic acid (KA) during early postnatal development impair neuronal and/or glial ciliogenesis. Mice received PTZ (50 or 100mg/kg), KA (2mg/kg), or saline either once at birth (P0), or once daily from P0 to P4. Using immunohistochemistry and electron microscopy, the cilia of neurons and glia were examined at P7, P14, and P42. A total of 83 regions were analyzed, representing 13 unique neocortical and hippocampal regions. Neuronal cilia were identified by co-expression of NeuN and type 3 adenylyl cyclase (ACIII) or somatostatin receptor 3 (SSTR3), while glial cilia were identified by co-expression of GFAP, Arl13b, and gamma-tubulin. We found that PTZ exposure at either P0 or from P0 to P4 induced convulsive behavior, followed by acute and lasting effects on neuronal cilia lengths that varied depending on the cortical region, PTZ dose, injection frequency, and time post-PTZ. Both increases and decreases in neuronal cilia length were observed. No changes in the length of glial cilia were observed under any of the test conditions. Lastly, we found that a single KA seizure at P0 led to similar abnormalities in neuronal cilia lengths. Our results suggest that seizure(s) occurring during early stages of cortical development induce persistent and widespread changes in neuronal cilia length. Given the impact neuronal cilia have on neuronal differentiation, ELS-induced changes in ciliogenesis may

  15. Attenuation of Magnesium Sulfate on CoCl₂-Induced Cell Death by Activating ERK1/2/MAPK and Inhibiting HIF-1α via Mitochondrial Apoptotic Signaling Suppression in a Neuronal Cell Line.

    PubMed

    Huang, Chih-Yang; Hsieh, You-Liang; Ju, Da-Tong; Lin, Chien-Chung; Kuo, Chia-Hua; Liou, Yi-Fan; Ho, Tsung-Jung; Tsai, Chang-Hai; Tsai, Fuu-Jen; Lin, Jing-Ying

    2015-08-31

    Magnesium sulfate (MgSO₄) ameliorates hypoxia/ischemia-induced neuronal apoptosis in a rat model. This study aimed to investigate the mechanisms governing the anti-apoptotic effect of MgSO₄ on cobalt chloride (CoCl₂)-exposed NB41A3 mouse neuroblastoma cells. MgSO₄ increased the viability of NB41A3 cells treated with CoCl₂ in a dose-dependent manner. MgSO₄ treatment was shown to lead to an increase in the anti-apoptotic Bcl-2 family proteins, with a concomitant decrease in the pro-apoptotic proteins. MgSO₄ also attenuated the CoCl₂-induced disruption of mitochondrial membrane potential (ΔΨ(m)) and reduced the release of cytochrome c form the mitochondria to the cytosol. Furthermore, exposure to CoCl₂ caused activation of the hypoxia-inducible factor 1α (HIF-1α). On the other hand, MgSO₄ markedly reduced CoCl₂-induced HIF-1α activation and suppressed HIF-1α downstream protein BNIP3. MgSO₄ treatment induced ERK1/2 activation and attenuated CoCl₂-induced activation of p38 and JNK. Addition of the ERK1/2 inhibitor U0126 significantly reduced the ability of MgSO₄ to protect neurons from CoCl₂-induced mitochondrial apoptotic events. However, incubation of cultures with the p38 and JNK inhibitors did not significantly affect MgSO₄-mediated neuroprotection. MgSO₄ appears to suppress CoCl₂-induced NB41A3 cell death by activating ERK1/2/ MAPK pathways, which further modulates the role of Bcl-2 family proteins and mitochondria in NB41A3 cells. Our data suggest that MgSO₄ may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways. PMID:26211648

  16. Transporting mitochondria in neurons

    PubMed Central

    Course, Meredith M.; Wang, Xinnan

    2016-01-01

    Neurons demand vast and vacillating supplies of energy. As the key contributors of this energy, as well as primary pools of calcium and signaling molecules, mitochondria must be where the neuron needs them, when the neuron needs them. The unique architecture and length of neurons, however, make them a complex system for mitochondria to navigate. To add to this difficulty, mitochondria are synthesized mainly in the soma, but must be transported as far as the distant terminals of the neuron. Similarly, damaged mitochondria—which can cause oxidative stress to the neuron—must fuse with healthy mitochondria to repair the damage, return all the way back to the soma for disposal, or be eliminated at the terminals. Increasing evidence suggests that the improper distribution of mitochondria in neurons can lead to neurodegenerative and neuropsychiatric disorders. Here, we will discuss the machinery and regulatory systems used to properly distribute mitochondria in neurons, and how this knowledge has been leveraged to better understand neurological dysfunction. PMID:27508065

  17. How microglia kill neurons.

    PubMed

    Brown, Guy C; Vilalta, Anna

    2015-12-01

    Microglia are resident brain macrophages that become inflammatory activated in most brain pathologies. Microglia normally protect neurons, but may accidentally kill neurons when attempting to limit infections or damage, and this may be more common with degenerative disease as there was no significant selection pressure on the aged brain in the past. A number of mechanisms by which activated microglia kill neurons have been identified, including: (i) stimulation of the phagocyte NADPH oxidase (PHOX) to produce superoxide and derivative oxidants, (ii) expression of inducible nitric oxide synthase (iNOS) producing NO and derivative oxidants, (iii) release of glutamate and glutaminase, (iv) release of TNFα, (v) release of cathepsin B, (vi) phagocytosis of stressed neurons, and (vii) decreased release of nutritive BDNF and IGF-1. PHOX stimulation contributes to microglial activation, but is not directly neurotoxic unless NO is present. NO is normally neuroprotective, but can react with superoxide to produce neurotoxic peroxynitrite, or in the presence of hypoxia inhibit mitochondrial respiration. Glutamate can be released by glia or neurons, but is neurotoxic only if the neurons are depolarised, for example as a result of mitochondrial inhibition. TNFα is normally neuroprotective, but can become toxic if caspase-8 or NF-κB activation are inhibited. If the above mechanisms do not kill neurons, they may still stress the neurons sufficiently to make them susceptible to phagocytosis by activated microglia. We review here whether microglial killing of neurons is an artefact, makes evolutionary sense or contributes in common neuropathologies and by what mechanisms. This article is part of a Special Issue entitled SI: Neuroprotection. PMID:26341532

  18. Gene co-expression analysis identifies brain regions and cell types involved in migraine pathophysiology: a GWAS-based study using the Allen Human Brain Atlas.

    PubMed

    Eising, Else; Huisman, Sjoerd M H; Mahfouz, Ahmed; Vijfhuizen, Lisanne S; Anttila, Verneri; Winsvold, Bendik S; Kurth, Tobias; Ikram, M Arfan; Freilinger, Tobias; Kaprio, Jaakko; Boomsma, Dorret I; van Duijn, Cornelia M; Järvelin, Marjo-Riitta R; Zwart, John-Anker; Quaye, Lydia; Strachan, David P; Kubisch, Christian; Dichgans, Martin; Davey Smith, George; Stefansson, Kari; Palotie, Aarno; Chasman, Daniel I; Ferrari, Michel D; Terwindt, Gisela M; de Vries, Boukje; Nyholt, Dale R; Lelieveldt, Boudewijn P F; van den Maagdenberg, Arn M J M; Reinders, Marcel J T

    2016-04-01

    Migraine is a common disabling neurovascular brain disorder typically characterised by attacks of severe headache and associated with autonomic and neurological symptoms. Migraine is caused by an interplay of genetic and environmental factors. Genome-wide association studies (GWAS) have identified over a dozen genetic loci associated with migraine. Here, we integrated migraine GWAS data with high-resolution spatial gene expression data of normal adult brains from the Allen Human Brain Atlas to identify specific brain regions and molecular pathways that are possibly involved in migraine pathophysiology. To this end, we used two complementary methods. In GWAS data from 23,285 migraine cases and 95,425 controls, we first studied modules of co-expressed genes that were calculated based on human brain expression data for enrichment of genes that showed association with migraine. Enrichment of a migraine GWAS signal was found for five modules that suggest involvement in migraine pathophysiology of: (i) neurotransmission, protein catabolism and mitochondria in the cortex; (ii) transcription regulation in the cortex and cerebellum; and (iii) oligodendrocytes and mitochondria in subcortical areas. Second, we used the high-confidence genes from the migraine GWAS as a basis to construct local migraine-related co-expression gene networks. Signatures of all brain regions and pathways that were prominent in the first method also surfaced in the second method, thus providing support that these brain regions and pathways are indeed involved in migraine pathophysiology. PMID:26899160

  19. Genome-Wide Expression of Transcriptomes and Their Co-Expression Pattern in Subtropical Maize (Zea mays L.) under Waterlogging Stress

    PubMed Central

    Thirunavukkarasu, Nepolean; Hossain, Firoz; Mohan, Sweta; Shiriga, Kaliyugam; Mittal, Swati; Sharma, Rinku; Singh, Rita Kumari; Gupta, Hari Shankar

    2013-01-01

    Waterlogging causes extensive damage to maize crops in tropical and subtropical regions. The identification of tolerance genes and their interactions at the molecular level will be helpful to engineer tolerant genotypes. A whole-genome transcriptome assay revealed the specific role of genes in response to waterlogging stress in susceptible and tolerant genotypes. Genes involved in the synthesis of ethylene and auxin, cell wall metabolism, activation of G-proteins and formation of aerenchyma and adventitious roots, were upregulated in the tolerant genotype. Many transcription factors, particularly ERFs, MYB, HSPs, MAPK, and LOB-domain protein were involved in regulation of these traits. Genes responsible for scavenging of ROS generated under stress were expressed along with those involved in carbohydrate metabolism. The physical locations of 21 genes expressed in the tolerant genotype were found to correspond with the marker intervals of known QTLs responsible for development of adaptive traits. Among the candidate genes, most showed synteny with genes of sorghum and foxtail millet. Co-expression analysis of 528 microarray samples including 16 samples from the present study generated seven functional modules each in the two genotypes, with differing characteristics. In the tolerant genotype, stress genes were co-expressed along with peroxidase and fermentation pathway genes. PMID:23936429

  20. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity. PMID:23624043

  1. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes

    PubMed Central

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  2. Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes.

    PubMed

    Abu Bakar, Fauziah; Yeo, Chew Chieng; Harikrishna, Jennifer Ann

    2016-01-01

    Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells. PMID:27104531

  3. Discovering gene re-ranking efficiency and conserved gene-gene relationships derived from gene co-expression network analysis on breast cancer data

    PubMed Central

    Bourdakou, Marilena M.; Athanasiadis, Emmanouil I.; Spyrou, George M.

    2016-01-01

    Systemic approaches are essential in the discovery of disease-specific genes, offering a different perspective and new tools on the analysis of several types of molecular relationships, such as gene co-expression or protein-protein interactions. However, due to lack of experimental information, this analysis is not fully applicable. The aim of this study is to reveal the multi-potent contribution of statistical network inference methods in highlighting significant genes and interactions. We have investigated the ability of statistical co-expression networks to highlight and prioritize genes for breast cancer subtypes and stages in terms of: (i) classification efficiency, (ii) gene network pattern conservation, (iii) indication of involved molecular mechanisms and (iv) systems level momentum to drug repurposing pipelines. We have found that statistical network inference methods are advantageous in gene prioritization, are capable to contribute to meaningful network signature discovery, give insights regarding the disease-related mechanisms and boost drug discovery pipelines from a systems point of view. PMID:26892392

  4. Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase.

    PubMed

    Sha, Chong; Yu, Xiao-Wei; Lin, Nai-Xin; Zhang, Meng; Xu, Yan

    2013-12-10

    Pichia pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, but there is still a large room of improvement for this expression system. Two factors drastically influence the lipase r27RCL production from Rhizopus chinensis CCTCC M201021, which are gene dosage and protein folding in the endoplasmic reticulum (ER). Regarding the effect of gene dosage, the enzyme activity for recombinant strain with three copies lipase gene was 1.95-fold higher than that for recombinant strain with only one copy lipase gene. In addition, the lipase production was further improved by co-expression with chaperone PDI involved in the disulfide bond formation in the ER. Overall, the maximum enzyme activity reached 355U/mL by the recombinant strain with one copy chaperone gene PDI plus five copies lipase gene proRCL in shaking flasks, which was 2.74-fold higher than that for the control strain with only one copy lipase gene. Overall, co-expression with PDI vastly increased the capacity for processing proteins of ER in P. pastoris. PMID:24315648

  5. Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

    PubMed Central

    Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

    2013-01-01

    Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

  6. Inhibition of 3' modification of small RNAs in virus-infected plants require spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins.

    PubMed

    Lózsa, Rita; Csorba, Tibor; Lakatos, Lóránt; Burgyán, József

    2008-07-01

    Plant viruses are inducers and targets of RNA silencing. Viruses counteract with RNA silencing by expressing silencing-suppressor proteins. Many of the identified proteins bind siRNAs, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. Here, we investigated the 3' modification of silencing-related small RNAs in Nicotiana benthamiana plants infected with viruses expressing RNA silencing suppressors, the p19 protein of Carnation Italian ringspot virus (CIRV) and HC-Pro of Tobacco etch virus (TEV). We found that CIRV had only a slight effect on viral siRNA 3' modification, but TEV significantly inhibited the 3' modification of si/miRNAs. We also found that p19 and HC-Pro were able to bind both 3' modified and non-modified small RNAs in vivo. The findings suggest that the 3' modification of viral siRNAs occurs in the cytoplasm, though miRNA 3' modification likely takes place in the nucleus as well. Both silencing suppressors inhibited the 3' modification of si/miRNAs when they and small RNAs were transiently co-expressed, suggesting that the inhibition of si/miRNA 3' modification requires spatial and temporal co-expression. Finally, our data revealed that a HEN1-like methyltransferase might account for the small RNA modification at the their 3'-terminal nucleotide in N. benthamiana. PMID:18539609

  7. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    SciTech Connect

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  8. Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

    PubMed Central

    Valton, Emeline; Amblard, Christian; Desmolles, François; Combourieu, Bruno; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-01-01

    In aquatic organisms, such as fish, blood is continually exposed to aquatic contaminants. Multidrug Resistance (MDR) proteins are ubiquitous detoxification membrane pumps, which recognize various xenobiotics. Moreover, their expression is induced by a large class of drugs and pollutants. We have highlighted the co-expression of a mini P-gp of 75 kDa and a P-gp of 140 kDa in the primary culture of brown trout erythrocytes and in the erythrocytes of wild brown trout collected from three rivers in the Auvergne region of France. In vitro experiments showed that benzo[a]pyrene, a highly toxic pollutant model, induced the co-expression of mini-P-gp and P-gp in trout erythrocytes in a dose-dependent manner and relay type response. Similarly, in the erythrocytes of wild brown trout collected from rivers contaminated by a mixture of PAH and other multi-residues of pesticides, mini-P-gp and P-gp were able to modulate their expression, according to the nature of the pollutants. The differential and complementary responses of mini-P-gp and P-gp in trout erythrocytes suggest the existence in blood cells of a real protective network against xenobiotics/drugs. This property could be exploited to develop a blood biomarker of river pollution. PMID:26854141

  9. Morphological, biophysical and synaptic properties of glutamatergic neurons of the mouse spinal dorsal horn

    PubMed Central

    Punnakkal, Pradeep; Schoultz, Carolin; Haenraets, Karen; Wildner, Hendrik; Zeilhofer, Hanns Ulrich

    2014-01-01

    Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one-third of all excitatory dorsal horn neurons and, as demonstrated by the co-expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell-type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after-hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin-induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent-evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory

  10. Neuronal Functions of ESCRTs

    PubMed Central

    Gao, Fen-Biao

    2012-01-01

    The endosomal sorting complexes required for transport (ESCRTs) regulate protein trafficking from endosomes to lysosomes. Recent studies have shown that ESCRTs are involved in various cellular processes, including membrane scission, microRNA function, viral budding, and the autophagy pathway in many tissues, including the nervous system. Indeed, dysfunctional ESCRTs are associated with neurodegeneration. However, it remains largely elusive how ESCRTs act in post-mitotic neurons, a highly specialized cell type that requires dynamic changes in neuronal structures and signaling for proper function. This review focuses on our current understandings of the functions of ESCRTs in neuronal morphology, synaptic plasticity, and neurodegenerative diseases. PMID:22438674

  11. Probing extracellular Sonic hedgehog in neurons

    PubMed Central

    Eitan, Erez; Petralia, Ronald S.; Wang, Ya-Xian; Indig, Fred E.; Mattson, Mark P.

    2016-01-01

    ABSTRACT The bioactivity of Sonic hedgehog (Shh) depends on specific lipid modifications; a palmitate at its N-terminus and a cholesterol at its C-terminus. This dual-lipid modification makes Shh molecules lipophilic, which prevents them from diffusing freely in extracellular space. Multiple lines of evidence indicate that Shh proteins are carried by various forms of extracellular vesicles (EVs). It also has been shown, for instance, that in some tissues Shh proteins are transported to neighboring cells directly via filopodia. We have previously reported that Shh proteins are expressed in hippocampal neurons. In this study we show that, in the hippocampus and cerebellum of postnatal day (P)2 rats, Shh is mostly found near or on the membrane surface of small neurites or filopodia. We also examined cultured hippocampal neurons where we observed noticeable and widespread Shh-immunolabeled vesicles located outside neurons. Through immunoelectron microscopy and biochemical analysis, we find Shh-containing EVs with a wide range of sizes. Unlike robust Shh activity in EVs isolated from cells overexpressing an N-terminal Shh fragment construct, we did not detect measurable Shh activity in EVs purified from the medium of cultured hippocampal neurons. These results suggest the complexity of the transcellular Shh signaling mechanisms in neurons. PMID:27387534

  12. Probing extracellular Sonic hedgehog in neurons.

    PubMed

    Eitan, Erez; Petralia, Ronald S; Wang, Ya-Xian; Indig, Fred E; Mattson, Mark P; Yao, Pamela J

    2016-01-01

    The bioactivity of Sonic hedgehog (Shh) depends on specific lipid modifications; a palmitate at its N-terminus and a cholesterol at its C-terminus. This dual-lipid modification makes Shh molecules lipophilic, which prevents them from diffusing freely in extracellular space. Multiple lines of evidence indicate that Shh proteins are carried by various forms of extracellular vesicles (EVs). It also has been shown, for instance, that in some tissues Shh proteins are transported to neighboring cells directly via filopodia. We have previously reported that Shh proteins are expressed in hippocampal neurons. In this study we show that, in the hippocampus and cerebellum of postnatal day (P)2 rats, Shh is mostly found near or on the membrane surface of small neurites or filopodia. We also examined cultured hippocampal neurons where we observed noticeable and widespread Shh-immunolabeled vesicles located outside neurons. Through immunoelectron microscopy and biochemical analysis, we find Shh-containing EVs with a wide range of sizes. Unlike robust Shh activity in EVs isolated from cells overexpressing an N-terminal Shh fragment construct, we did not detect measurable Shh activity in EVs purified from the medium of cultured hippocampal neurons. These results suggest the complexity of the transcellular Shh signaling mechanisms in neurons. PMID:27387534

  13. Selective IT neurons are selective along many dimensions.

    PubMed

    Zhivago, Kalathupiriyan A; Arun, S P

    2016-03-01

    Our visual abilities are unsurpassed because of a sophisticated code for objects located in the inferior temporal (IT) cortex. This code has remained a mystery because IT neurons show extremely diverse shape selectivity with no apparent organizing principle. Here, we show that there is an intrinsic component to selectivity in IT neurons. We tested IT neurons on distinct shapes and their parametric variations and asked whether neurons selective along one dimension were also selective along others. Selective neurons responded to fewer shapes and were narrowly tuned to local variations of these shapes, both along arbitrary morph lines and along variations in size, position, or orientation. For a subset of neurons, selective neurons were selective for both shape and texture. Finally, selective neurons were also more invariant in that they preserved their shape preferences across changes in size, position, and orientation. These observations indicate that there is an intrinsic constraint on the sharpness of tuning for the features coded by each IT neuron, making it always sharply tuned or always broadly tuned along all dimensions. We speculate that this may be an organizing principle throughout visual cortex. PMID:26823517

  14. Selective IT neurons are selective along many dimensions

    PubMed Central

    Zhivago, Kalathupiriyan A.

    2016-01-01

    Our visual abilities are unsurpassed because of a sophisticated code for objects located in the inferior temporal (IT) cortex. This code has remained a mystery because IT neurons show extremely diverse shape selectivity with no apparent organizing principle. Here, we show that there is an intrinsic component to selectivity in IT neurons. We tested IT neurons on distinct shapes and their parametric variations and asked whether neurons selective along one dimension were also selective along others. Selective neurons responded to fewer shapes and were narrowly tuned to local variations of these shapes, both along arbitrary morph lines and along variations in size, position, or orientation. For a subset of neurons, selective neurons were selective for both shape and texture. Finally, selective neurons were also more invariant in that they preserved their shape preferences across changes in size, position, and orientation. These observations indicate that there is an intrinsic constraint on the sharpness of tuning for the features coded by each IT neuron, making it always sharply tuned or always broadly tuned along all dimensions. We speculate that this may be an organizing principle throughout visual cortex. PMID:26823517

  15. Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding.

    PubMed

    Moret, Frédéric; Renaudot, Christelle; Bozon, Muriel; Castellani, Valérie

    2007-12-01

    Class III semaphorins (SemaIIIs) are intercellular cues secreted by surrounding tissues to guide migrating cells and axons in the developing organism. This chemotropic activity is crucial for the formation of nerves and vasculature. Intriguingly, SemaIIIs are also synthesized by neurons during axon pathfinding, but their function as intrinsic cues remains unknown. We have explored the role of Sema3A expression in motoneurons during spinal nerve development. Loss- and gain-of-function in the neural tube of the chick embryo were undertaken to target Sema3A expression in motoneurons while preserving Sema3A sources localized in peripheral tissues, known to provide important repulsive information for delineating the routes of motor axons towards their ventral or dorsal targets. Strikingly, Sema3A overexpression induced defasciculation and exuberant growth of motor axon projections into these normally non-permissive territories. Moreover, knockdown studies showed that motoneuronal Sema3A is required for correct spinal nerve compaction and dorsal motor axon extension. Further analysis of Sema3A gain- and loss-of-function in ex vivo models revealed that Sema3A in motoneurons sets the level of sensitivity of their growth cones to exogenous Sema3A exposure. This regulation is associated with post-transcriptional and local control of the availability of the Sema3A receptor neuropilin 1 at the growth cone surface. Thus, by modulating the strength of Sema3A-mediated environmental repulsive constraints, Sema3A in motoneurons enables axons to extend more or less far away from these repulsive sources. Such interplay between intrinsic and extrinsic Sema3A may represent a fundamental mechanism in the accurate specification of axon pathways. PMID:18039974

  16. Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons.

    PubMed

    Lee, Dong Woo; Cho, Pyung Sun; Lee, Han Kyu; Lee, Sang Hoon; Jung, Sung Jun; Oh, Seog Bae

    2015-10-01

    TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R-D5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca(2+) imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca(2+) responses via Ca(2+) influx through TRPV1 rather than Ca(2+) mobilization from intracellular Ca(2+) stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca(2+) response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca(2+) response. Taken together, our results suggest that the SKF-38393-induced Ca(2+) response results from the direct activation of TRPV1 by a PLC/DAG-mediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons. PMID:26319554

  17. Unique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking

    PubMed Central

    Fanous, Sanya; Guez-Barber, Danielle H; Goldart, Evan M; Schrama, Regina; Theberge, Florence RM; Shaham, Yavin; Hope, Bruce T

    2012-01-01

    Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6-h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell-sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial co-expression of NPY- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons. PMID:23113797

  18. Neuromorphic silicon neuron circuits.

    PubMed

    Indiveri, Giacomo; Linares-Barranco, Bernabé; Hamilton, Tara Julia; van Schaik, André; Etienne-Cummings, Ralph; Delbruck, Tobi; Liu, Shih-Chii; Dudek, Piotr; Häfliger, Philipp; Renaud, Sylvie; Schemmel, Johannes; Cauwenberghs, Gert; Arthur, John; Hynna, Kai; Folowosele, Fopefolu; Saighi, Sylvain; Serrano-Gotarredona, Teresa; Wijekoon, Jayawan; Wang, Yingxue; Boahen, Kwabena

    2011-01-01

    Hardware implementations of spiking neurons can be extremely useful for a large variety of applications, ranging from high-speed modeling of large-scale neural systems to real-time behaving systems, to bidirectional brain-machine interfaces. The specific circuit solutions used to implement silicon neurons depend on the application requirements. In this paper we describe the most common building blocks and techniques used to implement these circuits, and present an overview of a wide range of neuromorphic silicon neurons, which implement different computational models, ranging from biophysically realistic and conductance-based Hodgkin-Huxley models to bi-dimensional generalized adaptive integrate and fire models. We compare the different design methodologies used for each silicon neuron design described, and demonstrate their features with experimental results, measured from a wide range of fabricated VLSI chips. PMID:21747754

  19. Neuronal ubiquitin homeostasis

    PubMed Central

    Hallengren, Jada; Chen, Ping-Chung; Wilson, Scott M.

    2013-01-01

    Neurons have highly specialized intracellular compartments that facilitate the development and activity of the nervous system. Ubiquitination is a post-translational modification that controls many aspects of neuronal function by regulating protein abundance. Disruption of this signaling pathway has been demonstrated in neurological disorders such as Parkinson’s disease, Amyotrophic Lateral Sclerosis and Angleman Syndrome. Since many neurological disorders exhibit ubiquitinated protein aggregates, the loss of neuronal ubiquitin homeostasis may be an important contributor of disease. This review discusses the mechanisms utilized by neurons to control the free pool of ubiquitin necessary for normal nervous system development and function as well as new roles of protein ubiquitination in regulating synaptic activity. PMID:23686613

  20. Neuromorphic Silicon Neuron Circuits

    PubMed Central

    Indiveri, Giacomo; Linares-Barranco, Bernabé; Hamilton, Tara Julia; van Schaik, André; Etienne-Cummings, Ralph; Delbruck, Tobi; Liu, Shih-Chii; Dudek, Piotr; Häfliger, Philipp; Renaud, Sylvie; Schemmel, Johannes; Cauwenberghs, Gert; Arthur, John; Hynna, Kai; Folowosele, Fopefolu; Saighi, Sylvain; Serrano-Gotarredona, Teresa; Wijekoon, Jayawan; Wang, Yingxue; Boahen, Kwabena

    2011-01-01

    Hardware implementations of spiking neurons can be extremely useful for a large variety of applications, ranging from high-speed modeling of large-scale neural systems to real-time behaving systems, to bidirectional brain–machine interfaces. The specific circuit solutions used to implement silicon neurons depend on the application requirements. In this paper we describe the most common building blocks and techniques used to implement these circuits, and present an overview of a wide range of neuromorphic silicon neurons, which implement different computational models, ranging from biophysically realistic and conductance-based Hodgkin–Huxley models to bi-dimensional generalized adaptive integrate and fire models. We compare the different design methodologies used for each silicon neuron design described, and demonstrate their features with experimental results, measured from a wide range of fabricated VLSI chips. PMID:21747754

  1. Breadth of tuning in taste afferent neurons varies with stimulus strength.

    PubMed

    Wu, An; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2015-01-01

    Gustatory stimuli are detected by taste buds and transmitted to the hindbrain via sensory afferent neurons. Whether each taste quality (sweet, bitter and so on) is encoded by separate neurons ('labelled lines') remains controversial. We used mice expressing GCaMP3 in geniculate ganglion sensory neurons to investigate taste-evoked activity. Using confocal calcium imaging, we recorded responses to oral stimulation with prototypic taste stimuli. Up to 69% of neurons respond to multiple tastants. Moreover, neurons tuned to a single taste quality at low concentration become more broadly tuned when stimuli are presented at higher concentration. Responses to sucrose and monosodium glutamate are most related. Although mice prefer dilute NaCl solutions and avoid concentrated NaCl, we found no evidence for two separate populations of sensory neurons that encode this distinction. Altogether, our data suggest that taste is encoded by activity in patterns of peripheral sensory neurons and challenge the notion of strict labelled line coding. PMID:26373451

  2. Direct targeting of peptidergic amygdalar neurons by noradrenergic afferents: linking stress-integrative circuitry

    PubMed Central

    Kravets, J. L.; Reyes, B. A. S.; Unterwald, E. M.; Van Bockstaele, E. J.

    2014-01-01

    Amygdalar norepinephrine (NE) plays a key role in regulating neural responses to emotionally arousing stimuli and is involved in memory consolidation of emotionally charged events. Corticotropin-releasing factor (CRF) and dynorphin (DYN), two neuropeptides that mediate the physiological and behavioral responses to stress, are abundant in the central nucleus of the amygdala (CeA), and directly innervate brainstem noradrenergic locus coeruleus (LC) neurons. Whether the CRF- and DYN-containing amygdalar neurons receive direct noradrenergic innervation has not yet been elucidated. The present study sought to define cellular substrates underlying noradrenergic modulation of CRF- and DYN-containing neurons in the CeA using immunohistochemistry and electron microscopy. Ultrastructural analysis revealed that NE-labeled axon terminals form synapses with CRF- and DYN-containing neurons in the CeA. Semi-quantitative analysis showed that approximately 31% of NET-labeled axon terminals targeted CeA neurons that co-expressed DYN and CRF. As a major source of CRF innervation to the LC, it is also not known whether CRF-containing CeA neurons are directly targeted by noradrenergic afferents. To test this, retrograde tract-tracing using FluoroGold (FG) from the LC was combined with immunocytochemical detection of CRF and NET in the CeA. Our results revealed a population of LC-projecting CRF-containing CeA neurons that are directly innervated by NE afferents. Analysis showed that approximately 34% of NET-labeled axon terminals targeted LC-projecting CeA neurons that contain CRF. Taken together, these results indicate significant interactions between NE, CRF, and DYN in this critical limbic region and reveal direct synaptic interactions of NE with amygdalar CRF that influence the LC-NE arousal system. PMID:24271021

  3. Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons

    PubMed Central

    Ribeiro Fernandes, Hugo J.; Vowles, Jane; James, William S.; Cowley, Sally A.; Wade-Martins, Richard

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson’s disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. PMID:24586273

  4. Physiological characterisation of human iPS-derived dopaminergic neurons.

    PubMed

    Hartfield, Elizabeth M; Yamasaki-Mann, Michiko; Ribeiro Fernandes, Hugo J; Vowles, Jane; James, William S; Cowley, Sally A; Wade-Martins, Richard

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson's disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. PMID:24586273

  5. Co-Expression of Ezrin-CLIC5-Podocalyxin Is Associated with Migration and Invasiveness in Hepatocellular Carcinoma

    PubMed Central

    Flores-Téllez, Teresita N. J.; Lopez, Tania V.; Vásquez Garzón, Verónica Rocío; Villa-Treviño, Saúl

    2015-01-01

    Background and Aim Prognostic markers are important for predicting the progression and staging of hepatocellular carcinoma (HCC). Ezrin (EZR) and Podocalyxin (PODXL) are proteins associated with invasion, migration and poor prognosis in various types of cancer. Recently, it has been observed that chloride intracellular channel 5 (CLIC5) forms a complex with EZR and PODXL and that it is required for podocyte structure and function. In this study, we evaluated the overexpression of EZR, PODXL and CLIC5 in HCC. Methods The modified resistant hepatocyte model (MRHR), human biopsies and HCC cell lines (HepG2, Huh7 and SNU387) were used in this study. Gene and protein expression levels were evaluated in the MRHR by qRT-PCR, Western blot and immunohistochemistry analyses, and protein expression in the human biopsies was evaluated by immunohistochemistry. Protein expression in the HCC cell lines was evaluated by immunofluorescence and Western blot, also the migration and invasive abilities of Huh7 cells were evaluated using shRNA-mediated inhibition. Results Our results indicated that these genes and proteins were overexpressed in HCC. Moreover, when the expression of CLIC5 and PODXL was inhibited in Huh7 cells, we observed decreased migration and invasion. Conclusion This study suggested that EZR, CLIC5 and PODXL could be biological markers to predict the prognosis of HCC and that these proteins participate in migration and invasion processes. PMID:26135398

  6. NeuronBank: A Tool for Cataloging Neuronal Circuitry

    PubMed Central

    Katz, Paul S.; Calin-Jageman, Robert; Dhawan, Akshaye; Frederick, Chad; Guo, Shuman; Dissanayaka, Rasanjalee; Hiremath, Naveen; Ma, Wenjun; Shen, Xiuyn; Wang, Hsui C.; Yang, Hong; Prasad, Sushil; Sunderraman, Rajshekhar; Zhu, Ying

    2010-01-01

    The basic unit of any nervous system is the neuron. Therefore, understanding the operation of nervous systems ultimately requires an inventory of their constituent neurons and synaptic connectivity, which form neural circuits. The presence of uniquely identifiable neurons or classes of neurons in many invertebrates has facilitated the construction of cellular-level connectivity diagrams that can be generalized across individuals within a species. Homologous neurons can also be recognized across species. Here we describe NeuronBank.org, a web-based tool that we are developing for cataloging, searching, and analyzing neuronal circuitry within and across species. Information from a single species is represented in an individual branch of NeuronBank. Users can search within a branch or perform queries across branches to look for similarities in neuronal circuits across species. The branches allow for an extensible ontology so that additional characteristics can be added as knowledge grows. Each entry in NeuronBank generates a unique accession ID, allowing it to be easily cited. There is also an automatic link to a Wiki page allowing an encyclopedic explanation of the entry. All of the 44 previously published neurons plus one previously unpublished neuron from the mollusc, Tritonia diomedea, have been entered into a branch of NeuronBank as have 4 previously published neurons from the mollusc, Melibe leonina. The ability to organize information about neuronal circuits will make this information more accessible, ultimately aiding research on these important models. PMID:20428500

  7. Co-expression of TTF-1 and neuroendocrine markers in the human fetal lung and pulmonary neuroendocrine tumors.

    PubMed

    Miskovic, Josip; Brekalo, Zdrinko; Vukojevic, Katarina; Miskovic, Helena Radic; Kraljevic, Daniela; Todorovic, Jelena; Soljic, Violeta

    2015-01-01

    The expression pattern of thyroid transcription factor 1 (TTF-1) and neuroendocrine markers, neuron cell adhesion molecule (NCAM; CD56), chromogranin A (CgA) and synaptophysin (Syp), of different lung cell lineages was histologically analyzed in 15 normal human fetal lungs and 12 neuroendocrine tumors (NETs) using immunohistochemical methods. During pseudoglandular phase strong nuclear TTF-1 staining was detected in the columnar nonciliated epithelial cells, while NCAM, CgA and Syp had a moderate expression in the proximal airways and mild expression in the distal airways. Neuroendocrine cells (NECs) in proximal lung airway were co-localizing TTF-1 and other neuroendocrine markers while neuroendocrine bodies (NEBs) exhibit only staining with NCAM and Syp. In the canalicular phase TTF-1 nuclear staining was expressed only in several epithelial cells in proximal airways, while budding airways epithelium showed strong TTF-1 expression. Expression of NCAM, CgA and Syp in this phase equals the one in pseudoglandular phase. NEBs cells were co-localizing TTF-1 and NCAM in proximal airways and few NECs in distal airway were co-localizing TTF-1 and Syp. TTF-1 staining in the saccular phase was limited to subsets of epithelial cells in the proximal airways with stronger positivity in the distal airways. NCAM expression is moderate only in proximal airways, while Syp and CgA show mild expression in proximal and distal airways. NECs were co-localizing TTF-1 and NCAM in proximal lung airway. With regard to NECs, all small cell lung cancer (SCLC) cells had strong TTF-1, NCAM, Syp and CgA positivity and TTF-1 co-localized with other neuroendocrine markers. All pulmonary typical carcinoids were TTF-1 negative, while pulmonary atypical carcinoids were focal positive for TTF-1 and some neoplastic cells co-localized TTF-1 with neuroendocrine markers. Our results indicate that TTF-1 expression in NECs suggests a possible role in their normal development and differentiation. Our

  8. Interaction of co-expressed mu- and delta-opioid receptors in transfected rat pituitary GH(3) cells.

    PubMed

    Martin, N A; Prather, P L

    2001-04-01

    mu- and delta-Opioid agonists interact in a synergistic manner to produce analgesia in several animal models. Additionally, receptor binding studies using membranes derived from brain tissue indicate that interactions between mu- and delta-opioid receptors might be responsible for the observation of multiple opioid receptor subtypes. To examine potential interactions between mu- and delta-opioid receptors, we examined receptor binding and functional characteristics of mu-, delta-, or both mu- and delta-opioid receptors stably transfected in rat pituitary GH(3) cells (GH(3)MOR, GH(3)DOR, and GH(3)MORDOR, respectively). Saturation and competition binding experiments revealed that coexpression of mu- and delta-opioid receptors resulted in the appearance of multiple affinity states for mu- but not delta-opioid receptors. Additionally, coadministration of selective mu- and delta-opioid agonists in GH(3)MORDOR cells resulted in a synergistic competition with [(3)H][D-Pen(2,5)]enkephalin (DPDPE) for delta-opioid receptors. Finally, when equally effective concentrations of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) and two different delta-opioid agonists (DPDPE or 2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydroquinolino-[2,3,3-g]-isoquinoline; TAN67) were coadministered in GH(3)MORDOR cells, a synergistic inhibition of adenylyl cyclase activity was observed. These results strongly suggest that cotransfection of mu- and delta-opioid receptors alters the binding and functional characteristics of the receptors. Therefore, we propose that the simultaneous exposure of GH(3)MORDOR cells to selective mu- and delta-opioid agonists produces an interaction between receptors resulting in enhanced receptor binding. This effect is translated into an augmented ability of these agonists to inhibit adenylyl cyclase activity. Similar interactions occurring in neurons that express both mu- and delta-opioid receptors could explain observations of multiple

  9. Physiological Roles of Non-Neuronal NMDA Receptors.

    PubMed

    Hogan-Cann, Adam D; Anderson, Christopher M

    2016-09-01

    Glutamate serves as the dominant central nervous system (CNS) excitatory neurotransmitter, in part by activating N-methyl-D-aspartate receptors (NMDARs). While the structure, function, and distribution of neuronal NMDARs have been extensively elucidated, NMDARs are also expressed across a wide spectrum of non-neuronal cells, including central and peripheral glial cells, endothelium, kidney, bone, pancreas, and others. These receptors are poorly understood compared to neuronal receptors, but there is a developing consensus that they have distinct structural and functional properties when activated by glutamate, NMDAR co-agonists, and in some cases by metabolites of tryptophan and methionine. It is also clear that non-neuronal NMDARs may participate in an array of physiological and pathophysiological processes, including but not limited to bone deposition, wound healing, insulin secretion, blood-brain barrier integrity, and myelination. These developing lines of evidence are stimulating exploration of non-neuronal NMDARs as a therapeutic target in several disorders. PMID:27338838

  10. Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

    PubMed

    Xiao, Xiaolin; Moreno-Moral, Aida; Rotival, Maxime; Bottolo, Leonardo; Petretto, Enrico

    2014-01-01

    Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed

  11. A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory

    PubMed Central

    2013-01-01

    Background Moths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory. Results We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer’s yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 μg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that

  12. Multi-tissue Analysis of Co-expression Networks by Higher-Order Generalized Singular Value Decomposition Identifies Functionally Coherent Transcriptional Modules

    PubMed Central

    Bottolo, Leonardo; Petretto, Enrico

    2014-01-01

    Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed

  13. Model-based analysis of HER activation in cells co-expressing EGFR, HER2 and HER3.

    PubMed

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    2013-01-01

    The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches. PMID:23990774

  14. Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

    SciTech Connect

    Shankaran, Harish; Zhang, Yi; Tan, Yunbing; Resat, Haluk

    2013-08-22

    The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.

  15. Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

    PubMed

    Moroz, Leonid L; Kohn, Andrea B

    2015-12-01

    Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of

  16. Characterization of Kiss1 neurons using transgenic mouse models

    PubMed Central

    Cravo, Roberta M.; Margatho, Lisandra O.; Osborne-Lawrence, Sherri; Donato, José; Atkin, Stan; Bookout, Angie L.; Rovinsky, Sherry; Frazão, Renata; Lee, Charlotte E.; Gautron, Laurent; Zigman, Jeffrey M.; Elias, Carol F.

    2010-01-01

    Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptors (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or β-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed MC4R. Specifically in the preoptic area, 18–28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. PMID:21093546

  17. Neuronal avalanches and learning

    NASA Astrophysics Data System (ADS)

    de Arcangelis, Lucilla

    2011-05-01

    Networks of living neurons represent one of the most fascinating systems of biology. If the physical and chemical mechanisms at the basis of the functioning of a single neuron are quite well understood, the collective behaviour of a system of many neurons is an extremely intriguing subject. Crucial ingredient of this complex behaviour is the plasticity property of the network, namely the capacity to adapt and evolve depending on the level of activity. This plastic ability is believed, nowadays, to be at the basis of learning and memory in real brains. Spontaneous neuronal activity has recently shown features in common to other complex systems. Experimental data have, in fact, shown that electrical information propagates in a cortex slice via an avalanche mode. These avalanches are characterized by a power law distribution for the size and duration, features found in other problems in the context of the physics of complex systems and successful models have been developed to describe their behaviour. In this contribution we discuss a statistical mechanical model for the complex activity in a neuronal network. The model implements the main physiological properties of living neurons and is able to reproduce recent experimental results. Then, we discuss the learning abilities of this neuronal network. Learning occurs via plastic adaptation of synaptic strengths by a non-uniform negative feedback mechanism. The system is able to learn all the tested rules, in particular the exclusive OR (XOR) and a random rule with three inputs. The learning dynamics exhibits universal features as function of the strength of plastic adaptation. Any rule could be learned provided that the plastic adaptation is sufficiently slow.

  18. Rat Hepatocytes Weighted Gene Co-Expression Network Analysis Identifies Specific Modules and Hub Genes Related to Liver Regeneration after Partial Hepatectomy

    PubMed Central

    Zhou, Yun; Xu, Jiucheng; Liu, Yunqing; Li, Juntao; Chang, Cuifang; Xu, Cunshuan

    2014-01-01

    The recovery of liver mass is mainly mediated by proliferation of hepatocytes after 2/3 partial hepatectomy (PH) in rats. Studying the gene expression profiles of hepatocytes after 2/3 PH will be helpful to investigate the molecular mechanisms of liver regeneration (LR). We report here the first application of weighted gene co-expression network analysis (WGCNA) to analyze the biological implications of gene expression changes associated with LR. WGCNA identifies 12 specific gene modules and some hub genes from hepatocytes genome-scale microarray data in rat LR. The results suggest that upregulated MCM5 may promote hepatocytes proliferation during LR; BCL3 may play an important role by activating or inhibiting NF-kB pathway; MAPK9 may play a permissible role in DNA replication by p38 MAPK inactivation in hepatocytes proliferation stage. Thus, WGCNA can provide novel insight into understanding the molecular mechanisms of LR. PMID:24743545

  19. Engineering of Saccharomyces cerevisiae to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase, xylosidase and a bacterial xylose isomerase.

    PubMed

    Mert, Marlin John; la Grange, Daniël Coenrad; Rose, Shaunita Hellouise; van Zyl, Willem Heber

    2016-04-01

    Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes. PMID:26749525

  20. Mining the bladder cancer-associated genes by an integrated strategy for the construction and analysis of differential co-expression networks

    PubMed Central

    2015-01-01

    Background Bladder cancer is the most common malignant tumor of the urinary system and it is a heterogeneous disease with both superficial and invasive growth. However, its aetiological agent is still unclear. And it is indispensable to find key genes or modules causing the bladder cancer. Based on gene expression microarray datasets, constructing differential co-expression networks (DCNs) is an important method to investigate diseases and there have been some relevant good tools such as R package 'WGCNA', 'DCGL'. Results Employing an integrated strategy, 36 up-regulated differentially expressed genes (DEGs) and 356 down-regulated DEGs were selected and main functions of those DEGs are cellular physiological precess(24 up-regulated DEGs; 167 down-regulated DEGs) and cellular metabolism (19 up-regulated DEGs; 104 down-regulated DEGs). The up-regulated DEGs are mainly involved in the the pathways related to "metabolism". By comparing two DCNs between the normal and cancer states, we found some great changes in hub genes and topological structure, which suggest that the modules of two different DCNs change a lot. Especially, we screened some hub genes of a differential subnetwork between the normal and the cancer states and then do bioinformatics analysis for them. Conclusions Through constructing and analyzing two differential co-expression networks at different states using the screened DEGs, we found some hub genes associated with the bladder cancer. The results of the bioinformatics analysis for those hub genes will support the biological experiments and the further treatment of the bladder cancer. PMID:25707808

  1. Yeast gene CMR1/YDL156W is consistently co-expressed with genes participating in DNA-metabolic processes in a variety of stringent clustering experiments

    PubMed Central

    Abu-Jamous, Basel; Fa, Rui; Roberts, David J.; Nandi, Asoke K.

    2013-01-01

    The binarization of consensus partition matrices (Bi-CoPaM) method has, among its unique features, the ability to perform ensemble clustering over the same set of genes from multiple microarray datasets by using various clustering methods in order to generate tunable tight clusters. Therefore, we have used the Bi-CoPaM method to the most synchronized 500 cell-cycle-regulated yeast genes from different microarray datasets to produce four tight, specific and exclusive clusters of co-expressed genes. We found 19 genes formed the tightest of the four clusters and this included the gene CMR1/YDL156W, which was an uncharacterized gene at the time of our investigations. Two very recent proteomic and biochemical studies have independently revealed many facets of CMR1 protein, although the precise functions of the protein remain to be elucidated. Our computational results complement these biological results and add more evidence to their recent findings of CMR1 as potentially participating in many of the DNA-metabolism processes such as replication, repair and transcription. Interestingly, our results demonstrate the close co-expressions of CMR1 and the replication protein A (RPA), the cohesion complex and the DNA polymerases α, δ and ɛ, as well as suggest functional relationships between CMR1 and the respective proteins. In addition, the analysis provides further substantial evidence that the expression of the CMR1 gene could be regulated by the MBF complex. In summary, the application of a novel analytic technique in large biological datasets has provided supporting evidence for a gene of previously unknown function, further hypotheses to test, and a more general demonstration of the value of sophisticated methods to explore new large datasets now so readily generated in biological experiments. PMID:23349438

  2. Co-expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor-β1 into a biologically active protein.

    PubMed

    Wilbers, Ruud H P; Westerhof, Lotte B; van Raaij, Debbie R; van Adrichem, Marloes; Prakasa, Andreas D; Lozano-Torres, Jose L; Bakker, Jaap; Smant, Geert; Schots, Arjen

    2016-08-01

    Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing. PMID:26834022

  3. [Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis].

    PubMed

    Li, Wu; Deng, Guangcun; Liu, Xiaoming; Wang, Yujiong

    2014-02-01

    CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3'-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3'-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens. PMID:24941747

  4. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    SciTech Connect

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  5. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    PubMed

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  6. Kappe neurons, a novel population of olfactory sensory neurons

    PubMed Central

    Ahuja, Gaurav; Nia, Shahrzad Bozorg; Zapilko, Veronika; Shiriagin, Vladimir; Kowatschew, Daniel; Oka, Yuichiro; Korsching, Sigrun I.

    2014-01-01

    Perception of olfactory stimuli is mediated by distinct populations of olfactory sensory neurons, each with a characteristic set of morphological as well as functional parameters. Beyond two large populations of ciliated and microvillous neurons, a third population, crypt neurons, has been identified in teleost and cartilaginous fishes. We report here a novel, fourth olfactory sensory neuron population in zebrafish, which we named kappe neurons for their characteristic shape. Kappe neurons are identified by their Go-like immunoreactivity, and show a distinct spatial distribution within the olfactory epithelium, similar to, but significantly different from that of crypt neurons. Furthermore, kappe neurons project to a single identified target glomerulus within the olfactory bulb, mdg5 of the mediodorsal cluster, whereas crypt neurons are known to project exclusively to the mdg2 glomerulus. Kappe neurons are negative for established markers of ciliated, microvillous and crypt neurons, but appear to have microvilli. Kappe neurons constitute the fourth type of olfactory sensory neurons reported in teleost fishes and their existence suggests that encoding of olfactory stimuli may require a higher complexity than hitherto assumed already in the peripheral olfactory system. PMID:24509431

  7. Kappe neurons, a novel population of olfactory sensory neurons

    NASA Astrophysics Data System (ADS)

    Ahuja, Gaurav; Nia, Shahrzad Bozorg; Zapilko, Veronika; Shiriagin, Vladimir; Kowatschew, Daniel; Oka, Yuichiro; Korsching, Sigrun I.

    2014-02-01

    Perception of olfactory stimuli is mediated by distinct populations of olfactory sensory neurons, each with a characteristic set of morphological as well as functional parameters. Beyond two large populations of ciliated and microvillous neurons, a third population, crypt neurons, has been identified in teleost and cartilaginous fishes. We report here a novel, fourth olfactory sensory neuron population in zebrafish, which we named kappe neurons for their characteristic shape. Kappe neurons are identified by their Go-like immunoreactivity, and show a distinct spatial distribution within the olfactory epithelium, similar to, but significantly different from that of crypt neurons. Furthermore, kappe neurons project to a single identified target glomerulus within the olfactory bulb, mdg5 of the mediodorsal cluster, whereas crypt neurons are known to project exclusively to the mdg2 glomerulus. Kappe neurons are negative for established markers of ciliated, microvillous and crypt neurons, but appear to have microvilli. Kappe neurons constitute the fourth type of olfactory sensory neurons reported in teleost fishes and their existence suggests that encoding of olfactory stimuli may require a higher complexity than hitherto assumed already in the peripheral olfactory system.

  8. Imaging voltage in neurons

    PubMed Central

    Peterka, Darcy S.; Takahashi, Hiroto; Yuste, Rafael

    2011-01-01

    In the last decades, imaging membrane potential has become a fruitful approach to study neural circuits, especially in invertebrate preparations with large, resilient neurons. At the same time, particularly in mammalian preparations, voltage imaging methods suffer from poor signal to noise and secondary side effects, and they fall short of providing single-cell resolution when imaging of the activity of neuronal populations. As an introduction to these techniques, we briefly review different voltage imaging methods (including organic fluorophores, SHG chromophores, genetic indicators, hybrid, nanoparticles and intrinsic approaches), and illustrate some of their applications to neuronal biophysics and mammalian circuit analysis. We discuss their mechanisms of voltage sensitivity, from reorientation, electrochromic or electro-optical phenomena, to interaction among chromophores or membrane scattering, and highlight their advantages and shortcomings, commenting on the outlook for development of novel voltage imaging methods. PMID:21220095

  9. Josephson junction simulation of neurons

    NASA Astrophysics Data System (ADS)

    Crotty, Patrick; Schult, Dan; Segall, Ken

    2010-07-01

    With the goal of understanding the intricate behavior and dynamics of collections of neurons, we present superconducting circuits containing Josephson junctions that model biologically realistic neurons. These “Josephson junction neurons” reproduce many characteristic behaviors of biological neurons such as action potentials, refractory periods, and firing thresholds. They can be coupled together in ways that mimic electrical and chemical synapses. Using existing fabrication technologies, large interconnected networks of Josephson junction neurons would operate fully in parallel. They would be orders of magnitude faster than both traditional computer simulations and biological neural networks. Josephson junction neurons provide a new tool for exploring long-term large-scale dynamics for networks of neurons.

  10. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  11. Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

    PubMed

    Huizinga, Ruth; van der Star, Baukje J; Kipp, Markus; Jong, Rosa; Gerritsen, Wouter; Clarner, Tim; Puentes, Fabiola; Dijkstra, Christine D; van der Valk, Paul; Amor, Sandra

    2012-03-01

    Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood-borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well-characterized antibodies to axonal antigens, that axonal damage is associated with HLA-DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA-DR(+) cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF-L), present in white and gray matter, was observed in human microglia. The number of NF-L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF-L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF-L is taken up and degraded by the cell. In vivo, NF-L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA-DR(+) cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens. PMID:22161990

  12. Combining tissue extraction and off-line capillary electrophoresis matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for neuropeptide analysis in individual neuronal organs using 2,5-dihydroxybenzoic acid as a multi-functional agent.

    PubMed

    Wang, Junhua; Jiang, Xiaoyue; Sturm, Robert M; Li, Lingjun

    2009-11-20

    In this study we report an improved protocol that combines simplified sample preparation and micro-scale separation for mass spectrometric analysis of neuropeptides from individual neuroendocrine organs of crab Cancer borealis. A simple, one-step extraction method with commonly used matrix-assisted laser desorption/ionization (MALDI) matrix, 2,5-dihydroxybenzoic acid (DHB), in saturated aqueous solution, is employed for improved extraction of neuropeptides. Furthermore, a novel use of DHB as background electrolyte for capillary electrophoresis (CE) separation in the off-line coupling of CE to MALDI-Fourier transform mass spectrometric (FT-MS) detection is also explored. The new CE electrolyte exhibits full compatibility with MALDI-MS analysis of neuropeptides in that both the peptide extraction process and MALDI detection utilize DHB. In addition, enhanced resolving power and improved sensitivity are also observed for CE-MALDI-MS of peptide mixture analysis. Collectively, the use of DHB has simplified the extraction and reduced the sample loss by elimination of homogenizing, drying, and desalting processes. In the mean time, the concurrent use of DHB as CE separation buffer and subsequent MALDI matrix offers improved spectral quality by eliminating the interferences from typical CE electrolyte in MALDI detection. PMID:19473662

  13. Nanoresolution radiology of neurons

    SciTech Connect

    Wu, H.R.; Chen, S.T.; Chu, Y.S.; Conley, R.; Bouet, N.; Chien, C.C.; Chen, H.H.; Lin, C.H.; Tung, H.T.; Chen, Y.S.; Margaritondo, G.; Je, J.H.; Hwu, Y.

    2013-04-08

    We report recent advances in hard-x-ray optics - including record spatial resolution - and in staining techniques that enable synchrotron microradiology to produce neurobiology images of quality comparable to electron and visible microscopy. In addition, microradiology offers excellent penetration and effective three-dimensional detection as required for many neuron studies. Our tests include tomographic reconstruction based on projection image sets.

  14. Nanoresolution radiology of neurons

    SciTech Connect

    Wu, H. R.; Chen, S. T.; Chu, Y. S.; Conley, R.; Bouet, N.; Chien, C. C.; Chen, H. H.; Lin, C. H.; Tung, H. T.; Chen, Y. S.; Margaritondo, G.; Je, J. H.; Hwu, Y.

    2012-05-29

    We report recent advances in hard-x-ray optics—including record spatial resolution—and in staining techniques that enable synchrotron microradiology to produce neurobiology images of quality comparable to electron and visible microscopy. In addition, microradiology offers excellent penetration and effective three-dimensional detection as required for many neuron studies. Our tests include tomographic reconstruction based on projection image sets.

  15. Neuronal porosome lipidome

    PubMed Central

    Lewis, Kenneth T; Maddipati, Krishna R; Taatjes, Douglas J; Jena, Bhanu P

    2014-01-01

    Cup-shaped lipoprotein structures called porosomes are the universal secretory portals at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release intravesicular contents. In neurons, porosomes measure ∼15 nm and are comprised of nearly 40 proteins, among them SNAREs, ion channels, the Gαo G-protein and several structural proteins. Earlier studies report the interaction of specific lipids and their influence on SNAREs, ion channels and G-protein function. Our own studies demonstrate the requirement of cholesterol for the maintenance of neuronal porosome integrity, and the influence of lipids on SNARE complex assembly. In this study, to further understand the role of lipids on porosome structure-function, the lipid composition of isolated neuronal porosome was determined using mass spectrometry. Using lipid-binding assays, the affinity of porosome-associated syntaxin-1A to various lipids was determined. Our mass spectrometry results demonstrate the presence of phosphatidylinositol phosphates (PIP's) and phosphatidic acid (PA) among other lipids, and the enriched presence of ceramide (Cer), lysophosphatidylinositol phosphates (LPIP) and diacylglycerol (DAG). Lipid binding assays demonstrate the binding of neuronal porosome to cardiolipin, and confirm its association with PIP's and PA. The ability of exogenous PA to alter protein–protein interaction and neurotransmitter release is further demonstrated from the study. PMID:25224862

  16. Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

    PubMed Central

    Mak, Sally K.; Huang, Y. Anne; Iranmanesh, Shifteh; Vangipuram, Malini; Sundararajan, Ramya; Nguyen, Loan; Langston, J. William; Schüle, Birgitt

    2012-01-01

    Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage. PMID:22567022

  17. Tetracycline inducible gene manipulation in serotonergic neurons.

    PubMed

    Weber, Tillmann; Renzland, Insa; Baur, Max; Mönks, Simon; Herrmann, Elke; Huppert, Verena; Nürnberg, Frank; Schönig, Kai; Bartsch, Dusan

    2012-01-01

    The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a

  18. Epigenomic Landscapes Reflect Neuronal Diversity.

    PubMed

    Henikoff, Steven

    2015-06-17

    Epigenomic profiling of complex tissues obscures regulatory elements that distinguish one cell type from another. In this issue of Neuron, Mo et al. (2015) apply cell-type-specific profiling to mouse neuronal subtypes and discover an unprecedented level of neuronal diversity. PMID:26087157

  19. The impact of high co-expression of Sp1 and HIF1α on prognosis of patients with hepatocellular cancer

    PubMed Central

    LIU, LIANG; JI, PING; QU, NING; PU, WEI-LIN; JIANG, DAO-WEN; LIU, WEI-YAN; LI, YA-QI; SHI, RONG-LIANG

    2016-01-01

    Transcription factor specificity protein 1 (Sp1) and hypoxia-inducible factor 1α (HIF1α) serve vital roles in tumor growth and metastasis. The present study aimed to evaluate the impact of co-expression of Sp1 and HIF1α on the prognosis of patients with hepatocellular cancer (HCC) using The Cancer Genome Atlas (TCGA) database and to validate the association between the expression levels of Sp1/HIF1α in HCC specimens and patient survival using immunohistochemical analysis. A total of 214 eligible patients with HCC from TCGA database were collected for the study. The expression profile of Sp1 and HIF1α were obtained from the TCGA RNAseq database. Clinicopathological characteristics, including age, height, weight, gender, race, ethnicity, family cancer history, serum α-fetoprotein (AFP), surgical procedures and TNM stage were collected. The Cox proportional hazards regression model and Kaplan-Meier curves were used to assess the relative factors. Receiver operating characteristic (ROC) curves for cancer-specific survival (CSS) prediction were plotted to compare the prediction ability of expression of Sp1 and HIF1α and their co-expression. The location and expression of Sp1 and HIF1α in the HCC tissues were detected by immunohistochemistry (IHC) to verify the association between these two genes and CSS. The results demonstrated that the expressions of Sp1 and HIF1α were significantly increased in the succumbed group (P=0.001), compared with the surviving group. The CSS rates were 60.1% at 3 years (1,067 days), 35.8% at 5 years (1,823 days) and 9.5% at 10 years (3,528 days). Multivariate Cox regression analysis demonstrated that only the high expression levels of Sp1 and HIF1α (≥2×103) were independent predictors for cancer mortality, with P=0.001 and P=0.029, respectively. The area under the curve for the ROC was found to be higher using the combination testing for two genes (0.751) in predicting cancer mortality, compared to a single gene (0.632 for Sp1

  20. Human in vitro reporter model of neuronal development and early differentiation processes

    PubMed Central

    Couillard-Despres, Sebastien; Quehl, Eike; Altendorfer, Katrin; Karl, Claudia; Ploetz, Sonja; Bogdahn, Ulrich; Winkler, Juergen; Aigner, Ludwig

    2008-01-01

    Background During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop in vitro reporter models using doublecortin promoter sequences. Results Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression. Conclusion Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms. PMID:18312642

  1. Neuronal cell cycle: the neuron itself and its circumstances

    PubMed Central

    Frade, José M; Ovejero-Benito, María C

    2015-01-01

    Neurons are usually regarded as postmitotic cells that undergo apoptosis in response to cell cycle reactivation. Nevertheless, recent evidence indicates the existence of a defined developmental program that induces DNA replication in specific populations of neurons, which remain in a tetraploid state for the rest of their adult life. Similarly, de novo neuronal tetraploidization has also been described in the adult brain as an early hallmark of neurodegeneration. The aim of this review is to integrate these recent developments in the context of cell cycle regulation and apoptotic cell death in neurons. We conclude that a variety of mechanisms exists in neuronal cells for G1/S and G2/M checkpoint regulation. These mechanisms, which are connected with the apoptotic machinery, can be modulated by environmental signals and the neuronal phenotype itself, thus resulting in a variety of outcomes ranging from cell death at the G1/S checkpoint to full proliferation of differentiated neurons. PMID:25590687

  2. Detection of Temperature Difference in Neuronal Cells

    PubMed Central

    Tanimoto, Ryuichi; Hiraiwa, Takumi; Nakai, Yuichiro; Shindo, Yutaka; Oka, Kotaro; Hiroi, Noriko; Funahashi, Akira

    2016-01-01

    For a better understanding of the mechanisms behind cellular functions, quantification of the heterogeneity in an organism or cells is essential. Recently, the importance of quantifying temperature has been highlighted, as it correlates with biochemical reaction rates. Several methods for detecting intracellular temperature have recently been established. Here we develop a novel method for sensing temperature in living cells based on the imaging technique of fluorescence of quantum dots. We apply the method to quantify the temperature difference in a human derived neuronal cell line, SH-SY5Y. Our results show that temperatures in the cell body and neurites are different and thus suggest that inhomogeneous heat production and dissipation happen in a cell. We estimate that heterogeneous heat dissipation results from the characteristic shape of neuronal cells, which consist of several compartments formed with different surface-volume ratios. Inhomogeneous heat production is attributable to the localization of specific organelles as the heat source. PMID:26925874

  3. ESR1 Is Co-Expressed with Closely Adjacent Uncharacterised Genes Spanning a Breast Cancer Susceptibility Locus at 6q25.1

    PubMed Central

    Dunbier, Anita K.; Anderson, Helen; Ghazoui, Zara; Lopez-Knowles, Elena; Pancholi, Sunil; Ribas, Ricardo; Drury, Suzanne; Sidhu, Kally; Leary, Alexandra; Martin, Lesley-Ann; Dowsett, Mitch

    2011-01-01

    Approximately 80% of human breast carcinomas present as oestrogen receptor α-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs = 0.67, 0.64, and 0.55 respectively, FDR<1×10−7). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERα antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences