WorldWideScience

Sample records for cell type-specific visualization

  1. Layer- and cell-type-specific subthreshold and suprathreshold effects of long-term monocular deprivation in rat visual cortex.

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    Medini, Paolo

    2011-11-23

    Connectivity and dendritic properties are determinants of plasticity that are layer and cell-type specific in the neocortex. However, the impact of experience-dependent plasticity at the level of synaptic inputs and spike outputs remains unclear along vertical cortical microcircuits. Here I compared subthreshold and suprathreshold sensitivity to prolonged monocular deprivation (MD) in rat binocular visual cortex in layer 4 and layer 2/3 pyramids (4Ps and 2/3Ps) and in thick-tufted and nontufted layer 5 pyramids (5TPs and 5NPs), which innervate different extracortical targets. In normal rats, 5TPs and 2/3Ps are the most binocular in terms of synaptic inputs, and 5NPs are the least. Spike responses of all 5TPs were highly binocular, whereas those of 2/3Ps were dominated by either the contralateral or ipsilateral eye. MD dramatically shifted the ocular preference of 2/3Ps and 4Ps, mostly by depressing deprived-eye inputs. Plasticity was profoundly different in layer 5. The subthreshold ocular preference shift was sevenfold smaller in 5TPs because of smaller depression of deprived inputs combined with a generalized loss of responsiveness, and was undetectable in 5NPs. Despite their modest ocular dominance change, spike responses of 5TPs consistently lost their typically high binocularity during MD. The comparison of MD effects on 2/3Ps and 5TPs, the main affected output cells of vertical microcircuits, indicated that subthreshold plasticity is not uniquely determined by the initial degree of input binocularity. The data raise the question of whether 5TPs are driven solely by 2/3Ps during MD. The different suprathreshold plasticity of the two cell populations could underlie distinct functional deficits in amblyopia.

  2. Cell-Type-Specific Optogenetics in Monkeys.

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    Namboodiri, Vijay Mohan K; Stuber, Garret D

    2016-09-08

    The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys.

  3. High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants

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    van der Winden Johannes

    2010-01-01

    Full Text Available Abstract Background Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells. Results We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B. Conclusions The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending

  4. The selection and function of cell type-specific enhancers.

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    Heinz, Sven; Romanoski, Casey E; Benner, Christopher; Glass, Christopher K

    2015-03-01

    The human body contains several hundred cell types, all of which share the same genome. In metazoans, much of the regulatory code that drives cell type-specific gene expression is located in distal elements called enhancers. Although mammalian genomes contain millions of potential enhancers, only a small subset of them is active in a given cell type. Cell type-specific enhancer selection involves the binding of lineage-determining transcription factors that prime enhancers. Signal-dependent transcription factors bind to primed enhancers, which enables these broadly expressed factors to regulate gene expression in a cell type-specific manner. The expression of genes that specify cell type identity and function is associated with densely spaced clusters of active enhancers known as super-enhancers. The functions of enhancers and super-enhancers are influenced by, and affect, higher-order genomic organization.

  5. Freedom of expression: cell-type-specific gene profiling.

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    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  6. Cell-type specific four-component hydrogel.

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    Timo Aberle

    Full Text Available In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin to generate a blend (technical term: quattroGel, an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

  7. Cell type-specific neuroprotective activity of untranslocated prion protein.

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    Elena Restelli

    Full Text Available BACKGROUND: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. PRINCIPAL FINDINGS: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. SIGNIFICANCE: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

  8. General approach for in vivo recovery of cell type-specific effector gene sets.

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    Barsi, Julius C; Tu, Qiang; Davidson, Eric H

    2014-05-01

    Differentially expressed, cell type-specific effector gene sets hold the key to multiple important problems in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various practical applications. Although individual cell types of interest have been recovered by various methods and analyzed, systematic recovery of multiple cell type-specific gene sets from whole developing organisms has remained problematic. Here we describe a general methodology using the sea urchin embryo, a material of choice because of the large-scale GRNs already solved for this model system. This method utilizes the regulatory states expressed by given cells of the embryo to define cell type and includes a fluorescence activated cell sorting (FACS) procedure that results in no perturbation of transcript representation. We have extensively validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated embryonic skeletogenic cells and as a consequence, generated a prototypical cell type-specific transcriptome database.

  9. Cell-Type Specific Roles for PTEN in Establishing a Functional Retinal Architecture

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    Cantrup, Robert; Dixit, Rajiv; Palmesino, Elena; Bonfield, Stephan; Shaker, Tarek; Tachibana, Nobuhiko; Zinyk, Dawn; Dalesman, Sarah; Yamakawa, Kazuhiro; Stell, William K.; Wong, Rachel O.; Reese, Benjamin E.; Kania, Artur; Sauvé, Yves; Schuurmans, Carol

    2012-01-01

    Background The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture. Methodology/Principal Findings In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam. Conclusions/Significance We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular

  10. Cell-type specific roles for PTEN in establishing a functional retinal architecture.

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    Robert Cantrup

    Full Text Available BACKGROUND: The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture. METHODOLOGY/PRINCIPAL FINDINGS: In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam. CONCLUSIONS/SIGNIFICANCE: We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected

  11. Cell type-specific bipolar cell input to ganglion cells in the mouse retina.

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    Neumann, S; Hüser, L; Ondreka, K; Auler, N; Haverkamp, S

    2016-03-01

    Many distinct ganglion cell types, which are the output elements of the retina, were found to encode for specific features of a visual scene such as contrast, color information or movement. The detailed composition of retinal circuits leading to this tuning of retinal ganglion cells, however, is apart from some prominent examples, largely unknown. Here we aimed to investigate if ganglion cell types in the mouse retina receive selective input from specific bipolar cell types or if they sample their synaptic input non-selectively from all bipolar cell types stratifying within their dendritic tree. To address this question we took an anatomical approach and immunolabeled retinae of two transgenic mouse lines (GFP-O and JAM-B) with markers for ribbon synapses and type 2 bipolar cells. We morphologically identified all green fluorescent protein (GFP)-expressing ganglion cell types, which co-stratified with type 2 bipolar cells and assessed the total number of bipolar input synapses and the proportion of synapses deriving from type 2 bipolar cells. Only JAM-B ganglion cells received synaptic input preferentially from bipolar cell types other than type 2 bipolar cells whereas the other analyzed ganglion cell types sampled their bipolar input most likely from all bipolar cell terminals within their dendritic arbor.

  12. Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles.

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    Greulich, C; Diendorf, J; Gessmann, J; Simon, T; Habijan, T; Eggeler, G; Schildhauer, T A; Epple, M; Köller, M

    2011-09-01

    Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.

  13. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

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    Cremer Thomas

    2005-12-01

    Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

  14. Towards identifying host cell-type specific response patterns to bacterial endosymbiosis

    DEFF Research Database (Denmark)

    Gavrilovic, Srdjan

    of view, available techniques have relied heavily on whole organ analyses that disregard specificities of individual cell types. To address this issue we aimed to develop a technology for comparative global analysis of mature mRNA and small RNA populations at the cell type specific level in the model...... plant Lotus japonicus. A powerful approach referred to here as Defined Expression and RNA Affinity co-Purification (DERAP) was developed to study gene expression and small RNA populations in the host roots during early phases of signal exchange at the cell-type level. As a basis for DERAP analysis...

  15. Cell-type-specific, Aptamer-functionalized Agents for Targeted Disease Therapy.

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    Zhou, Jiehua; Rossi, John J

    2014-06-17

    One hundred years ago, Dr. Paul Ehrlich popularized the "magic bullet" concept for cancer therapy in which an ideal therapeutic agent would only kill the specific tumor cells it targeted. Since then, "targeted therapy" that specifically targets the molecular defects responsible for a patient's condition has become a long-standing goal for treating human disease. However, safe and efficient drug delivery during the treatment of cancer and infectious disease remains a major challenge for clinical translation and the development of new therapies. The advent of SELEX technology has inspired many groundbreaking studies that successfully adapted cell-specific aptamers for targeted delivery of active drug substances in both in vitro and in vivo models. By covalently linking or physically functionalizing the cell-specific aptamers with therapeutic agents, such as siRNA, microRNA, chemotherapeutics or toxins, or delivery vehicles, such as organic or inorganic nanocarriers, the targeted cells and tissues can be specifically recognized and the therapeutic compounds internalized, thereby improving the local concentration of the drug and its therapeutic efficacy. Currently, many cell-type-specific aptamers have been developed that can target distinct diseases or tissues in a cell-type-specific manner. In this review, we discuss recent advances in the use of cell-specific aptamers for targeted disease therapy, as well as conjugation strategies and challenges.

  16. Transition to chaos in random networks with cell-type-specific connectivity

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    Aljadeff, Johnatan; Stern, Merav; Sharpee, Tatyana

    2015-01-01

    In neural circuits, statistical connectivity rules strongly depend on cell-type identity. We study dynamics of neural networks with cell-type specific connectivity by extending the dynamic mean field method, and find that these networks exhibit a phase transition between silent and chaotic activity. By analyzing the locus of this transition, we derive a new result in random matrix theory: the spectral radius of a random connectivity matrix with block-structured variances. We apply our results to show how a small group of hyper-excitable neurons within the network can significantly increase the network’s computational capacity by bringing it into the chaotic regime. PMID:25768781

  17. Cell-type specific DNA methylation patterns define human breast cellular identity.

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    Petr Novak

    Full Text Available DNA methylation plays a role in a variety of biological processes including embryonic development, imprinting, X-chromosome inactivation, and stem cell differentiation. Tissue specific differential methylation has also been well characterized. We sought to extend these studies to create a map of differential DNA methylation between different cell types derived from a single tissue. Using three pairs of isogenic human mammary epithelial and fibroblast cells, promoter region DNA methylation was characterized using MeDIP coupled to microarray analysis. Comparison of DNA methylation between these cell types revealed nearly three thousand cell-type specific differentially methylated regions (ctDMRs. MassARRAY was performed upon 87 ctDMRs to confirm and quantify differential DNA methylation. Each of the examined regions exhibited statistically significant differences ranging from 10-70%. Gene ontology analysis revealed the overrepresentation of many transcription factors involved in developmental processes. Additionally, we have shown that ctDMRs are associated with histone related epigenetic marks and are often aberrantly methylated in breast cancer. Overall, our data suggest that there are thousands of ctDMRs which consistently exhibit differential DNA methylation and may underlie cell type specificity in human breast tissue. In addition, we describe the pathways affected by these differences and provide insight into the molecular mechanisms and physiological overlap between normal cellular differentiation and breast carcinogenesis.

  18. Innate immune response to pulmonary contusion: identification of cell type-specific inflammatory responses.

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    Hoth, J Jason; Wells, Jonathan D; Yoza, Barbara K; McCall, Charles E

    2012-04-01

    Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma, such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety of inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll-like receptors 2 and 4 (TLR2 and TLR4) mediate the inflammatory response to lung injury. In this study, we used chimeric mice generated by adoptive bone marrow transfer between TLR2 or TLR4 and wild-type mice. We found that, in the lung, both bone marrow-derived and nonmyeloid cells contribute to TLR-dependent inflammatory responses after injury in a cell type-specific manner. We also show a novel TLR2-dependent injury mechanism that is associated with enhanced airway epithelial cell apoptosis and increased pulmonary FasL and Fas expression in the lungs from injured mice. Thus, in addition to cardiopulmonary physiological dysfunction, cell type-specific TLR and their differential response to injury may provide novel specific targets for management of patients with pulmonary contusion.

  19. VARIATION ANALYSIS OF HPV16 CELL-TYPE-SPECIFIC ENHANCER IN CERVICAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Human papillomavirus16type(HPV16)ishighly associated with cervical carcinoma.Sometransfor mation genes in high-risk HPV genomeplayed ani mportant role[1].The E6and E7genes inHPV16can over-express intransfor mepithelial cellsand viral early promoter P97controls the expressionof E6/E7genes.Long control region(LCR)inHPV16genome induces the activity of P97.Thereexits cell-type-specific enhancer(CTSE)in LCRand there are many cellar factors specific bindingsites in CTSE such as NF1,AP1,TEF-2,whichbindspecifically...

  20. Ligation-free ribosome profiling of cell type-specific translation in the brain.

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    Hornstein, Nicholas; Torres, Daniela; Das Sharma, Sohani; Tang, Guomei; Canoll, Peter; Sims, Peter A

    2016-01-01

    Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.

  1. Systematic and Cell Type-Specific Telomere Length Changes in Subsets of Lymphocytes

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    Jue Lin

    2016-01-01

    Full Text Available Telomeres, the protective DNA-protein complexes at the ends of linear chromosomes, are important for genome stability. Leukocyte or peripheral blood mononuclear cell (PBMC telomere length is a potential biomarker for human aging that integrates genetic, environmental, and lifestyle factors and is associated with mortality and risks for major diseases. However, only a limited number of studies have examined longitudinal changes of telomere length and few have reported data on sorted circulating immune cells. We examined the average telomere length (TL in CD4+, CD8+CD28+, and CD8+CD28− T cells, B cells, and PBMCs, cross-sectionally and longitudinally, in a cohort of premenopausal women. We report that TL changes over 18 months were correlated among these three T cell types within the same participant. Additionally, PBMC TL change was also correlated with those of all three T cell types, and B cells. The rate of shortening for B cells was significantly greater than for the three T cell types. CD8+CD28− cells, despite having the shortest TL, showed significantly more rapid attrition when compared to CD8+CD28+ T cells. These results suggest systematically coordinated, yet cell type-specific responses to factors and pathways contribute to telomere length regulation.

  2. Systematic and Cell Type-Specific Telomere Length Changes in Subsets of Lymphocytes.

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    Lin, Jue; Cheon, Joshua; Brown, Rashida; Coccia, Michael; Puterman, Eli; Aschbacher, Kirstin; Sinclair, Elizabeth; Epel, Elissa; Blackburn, Elizabeth H

    2016-01-01

    Telomeres, the protective DNA-protein complexes at the ends of linear chromosomes, are important for genome stability. Leukocyte or peripheral blood mononuclear cell (PBMC) telomere length is a potential biomarker for human aging that integrates genetic, environmental, and lifestyle factors and is associated with mortality and risks for major diseases. However, only a limited number of studies have examined longitudinal changes of telomere length and few have reported data on sorted circulating immune cells. We examined the average telomere length (TL) in CD4+, CD8+CD28+, and CD8+CD28- T cells, B cells, and PBMCs, cross-sectionally and longitudinally, in a cohort of premenopausal women. We report that TL changes over 18 months were correlated among these three T cell types within the same participant. Additionally, PBMC TL change was also correlated with those of all three T cell types, and B cells. The rate of shortening for B cells was significantly greater than for the three T cell types. CD8+CD28- cells, despite having the shortest TL, showed significantly more rapid attrition when compared to CD8+CD28+ T cells. These results suggest systematically coordinated, yet cell type-specific responses to factors and pathways contribute to telomere length regulation.

  3. Cell type-specific glycosylation of Orai1 modulates store-operated Ca2+ entry.

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    Dörr, Kathrin; Kilch, Tatiana; Kappel, Sven; Alansary, Dalia; Schwär, Gertrud; Niemeyer, Barbara A; Peinelt, Christine

    2016-03-08

    N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca(2+) entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca(2+) sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca(2+) channels in the plasma membrane mediate Ca(2+) influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid-binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type-specific. Ca(2+) imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6-linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca(2+) entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type-specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca(2+) signaling and, thus, may contribute to pathophysiological Ca(2+) signaling observed in immune disease and cancer.

  4. Cell type-specific synaptic dynamics of synchronized bursting in the juvenile CA3 rat hippocampus.

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    Aradi, Ildiko; Maccaferri, Gianmaria

    2004-10-27

    Spontaneous synchronous bursting of the CA3 hippocampus in vitro is a widely studied model of physiological and pathological network synchronization. The role of inhibitory conductances during network bursting is not understood in detail, despite the fact that several antiepileptic drugs target GABA(A) receptors. Here, we show that the first manifestation of a burst event is a cell type-specific flurry of GABA(A) receptor-mediated inhibitory input to pyramidal cells, but not to stratum oriens horizontal interneurons. Moreover, GABA(A) receptor-mediated synaptic input is proportionally smaller in these interneurons compared with pyramidal cells. Computational models and dynamic-clamp studies using experimentally derived conductance waveforms indicate that both these factors modulate spike timing during synchronized activity. In particular, the different kinetics and the larger strength of GABAergic input to pyramidal cells defer action potential initiation and contribute to the observed delay of firing, so that the interneuronal activity leads the burst cycle. In contrast, excitatory inputs to both neuronal populations during a burst are kinetically similar, as required to maintain synchronicity. We also show that the natural pattern of activation of inhibitory and excitatory conductances during a synchronized burst cycle is different within the same neuronal population. In particular, GABA(A) receptor-mediated currents activate earlier and outlast the excitatory components driving the bursts. Thus, cell type-specific balance and timing of GABA(A) receptor-mediated input are critical to set the appropriate spike timing in pyramidal cells and interneurons and coordinate additional neurotransmitter release modulating burst strength and network frequency.

  5. The female gametophyte: an emerging model for cell type-specific systems biology in plant development

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    Marc William Schmid

    2015-11-01

    Full Text Available Systems biology, a holistic approach describing a system emerging from the interactions of its molecular components, critically depends on accurate qualitative determination and quantitative measurements of these components. Development and improvement of large-scale profiling methods (omics now facilitates comprehensive measurements of many relevant molecules. For multicellular organisms, such as animals, fungi, algae, and plants, the complexity of the system is augmented by the presence of specialized cell types and organs, and a complex interplay within and between them. Cell type-specific analyses are therefore crucial for the understanding of developmental processes and environmental responses. This review first gives an overview of current methods used for large-scale profiling of specific cell types exemplified by recent advances in plant biology. The focus then lies on suitable model systems to study plant development and cell type specification. We introduce the female gametophyte of flowering plants as an ideal model to study fundamental developmental processes. Moreover, the female reproductive lineage is of importance for the emergence of evolutionary novelties such as an unequal parental contribution to the tissue nurturing the embryo or the clonal production of seeds by asexual reproduction (apomixis. Understanding these processes is not only interesting from a developmental or evolutionary perspective, but bears great potential for further crop improvement and the simplification of breeding efforts. We finally highlight novel methods, which are already available or which will likely soon facilitate large-scale profiling of the specific cell types of the female gametophyte in both model and non-model species. We conclude that it may take only few years until an evolutionary systems biology approach toward female gametogenesis may decipher some of its biologically most interesting and economically most valuable processes.

  6. Cell type-specific translational repression of Cyclin B during meiosis in males.

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    Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

    2015-10-01

    The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.

  7. Analysis of cell-type-specific gene expression during mouse spermatogenesis

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Nielsen, John E; Hansen, Martin Asser

    2004-01-01

    In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume....... Combining results from these techniques allows determination of the cell-type-specific gene-expression patterns of many genes during spermatogenesis. Differential display was used to determine expression profiles with high sensitivity and independent of prior knowledge of the sequence, whereas DNA arrays...... quickly assess the expression profiles of all the genes. This identified three groups of gene-expression profiles. The major group corresponds to genes that are upregulated in spermatocytes during either the mid- or late- pachytene phase of spermatogenesis (stages VII-XI). This pachytene cluster...

  8. VARIATION ANALYSIS OF HPV16 CELL-TYPE-SPECIFIC ENHANCER IN CERVICAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    Liu Wenkang; Chu Yonglie; Ma Tianyou; Yang E; Cao Chunxia

    2006-01-01

    Objective To investigate the cell-type-specific enhancer (CTSE) in HPV16 and its variation in cervical carcinoma. Methods CTSEs were detected by polymerase chain reaction (PCR) in 58 cervical carcinoma from Shaanxi province; in addition variation of CTSEs was analyzed through single-strand conformation polymorphisms (SSCP). Results HPV16 CTSEs were detectable in 34 of 58 (57%) specimens and mutant rate was 41%(14/34) and the main mutations of chosen randomly variant CTSE (CTSEv) happened at YY1 binding sites in addition to glucocoticoid response elements (GRE). Conclusion CTSE in some specimens of Shaanxi province was obviously different from that in HPV16 wild type and variant CTSE might affect the transcriptional regulation of LCR on viral P97, which regulates over-expression of viral oncogenes in cervical carcinoma.

  9. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  10. Species- and cell type-specific interactions between CD47 and human SIRPalpha.

    Science.gov (United States)

    Subramanian, Shyamsundar; Parthasarathy, Ranganath; Sen, Shamik; Boder, Eric T; Discher, Dennis E

    2006-03-15

    CD47 on red blood cells (RBCs) reportedly signals "self" by binding SIRPalpha on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRPalpha1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRPalpha1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRPalpha-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRPalpha1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRPalpha1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRPalpha1 significantly. The results thus demonstrate that SIRPalpha-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity.

  11. Epigenetic regulation of normal human mammary cell type-specific miRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

    2011-08-26

    Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

  12. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  13. Partitioning Heritability of Regulatory and Cell-Type-Specific Variants across 11 Common Diseases

    Science.gov (United States)

    Gusev, Alexander; Lee, S. Hong; Trynka, Gosia; Finucane, Hilary; Vilhjálmsson, Bjarni J.; Xu, Han; Zang, Chongzhi; Ripke, Stephan; Bulik-Sullivan, Brendan; Stahl, Eli; Ripke, Stephan; Neale, Benjamin M.; Corvin, Aiden; Walters, James T.R.; Farh, Kai-How; Holmans, Peter A.; Lee, Phil; Bulik-Sullivan, Brendan; Collier, David A.; Huang, Hailiang; Pers, Tune H.; Agartz, Ingrid; Agerbo, Esben; Albus, Margot; Alexander, Madeline; Amin, Farooq; Bacanu, Silviu A.; Begemann, Martin; Belliveau, Richard A.; Bene, Judit; Bergen, Sarah E.; Bevilacqua, Elizabeth; Bigdeli, Tim B.; Black, Donald W.; Børglum, Anders D.; Bruggeman, Richard; Buccola, Nancy G.; Buckner, Randy L.; Byerley, William; Cahn, Wiepke; Cai, Guiqing; Campion, Dominique; Cantor, Rita M.; Carr, Vaughan J.; Carrera, Noa; Catts, Stanley V.; Chambert, Kimberly D.; Chan, Raymond C.K.; Chen, Ronald Y.L.; Chen, Eric Y.H.; Cheng, Wei; Cheung, Eric F.C.; Chong, Siow Ann; Cloninger, C. Robert; Cohen, David; Cohen, Nadine; Cormican, Paul; Craddock, Nick; Crowley, James J.; Curtis, David; Davidson, Michael; Davis, Kenneth L.; Degenhardt, Franziska; Del Favero, Jurgen; DeLisi, Lynn E.; Demontis, Ditte; Dikeos, Dimitris; Dinan, Timothy; Djurovic, Srdjan; Donohoe, Gary; Drapeau, Elodie; Duan, Jubao; Dudbridge, Frank; Durmishi, Naser; Eichhammer, Peter; Eriksson, Johan; Escott-Price, Valentina; Essioux, Laurent; Fanous, Ayman H.; Farrell, Martilias S.; Frank, Josef; Franke, Lude; Freedman, Robert; Freimer, Nelson B.; Friedl, Marion; Friedman, Joseph I.; Fromer, Menachem; Genovese, Giulio; Georgieva, Lyudmila; Gershon, Elliot S.; Giegling, Ina; Giusti-Rodrguez, Paola; Godard, Stephanie; Goldstein, Jacqueline I.; Golimbet, Vera; Gopal, Srihari; Gratten, Jacob; Grove, Jakob; de Haan, Lieuwe; Hammer, Christian; Hamshere, Marian L.; Hansen, Mark; Hansen, Thomas; Haroutunian, Vahram; Hartmann, Annette M.; Henskens, Frans A.; Herms, Stefan; Hirschhorn, Joel N.; Hoffmann, Per; Hofman, Andrea; Hollegaard, Mads V.; Hougaard, David M.; Ikeda, Masashi; Joa, Inge; Julià, Antonio; Kahn, René S.; Kalaydjieva, Luba; Karachanak-Yankova, Sena; Karjalainen, Juha; Kavanagh, David; Keller, Matthew C.; Kelly, Brian J.; Kennedy, James L.; Khrunin, Andrey; Kim, Yunjung; Klovins, Janis; Knowles, James A.; Konte, Bettina; Kucinskas, Vaidutis; Kucinskiene, Zita Ausrele; Kuzelova-Ptackova, Hana; Kähler, Anna K.; Laurent, Claudine; Keong, Jimmy Lee Chee; Lee, S. Hong; Legge, Sophie E.; Lerer, Bernard; Li, Miaoxin; Li, Tao; Liang, Kung-Yee; Lieberman, Jeffrey; Limborska, Svetlana; Loughland, Carmel M.; Lubinski, Jan; Lnnqvist, Jouko; Macek, Milan; Magnusson, Patrik K.E.; Maher, Brion S.; Maier, Wolfgang; Mallet, Jacques; Marsal, Sara; Mattheisen, Manuel; Mattingsdal, Morten; McCarley, Robert W.; McDonald, Colm; McIntosh, Andrew M.; Meier, Sandra; Meijer, Carin J.; Melegh, Bela; Melle, Ingrid; Mesholam-Gately, Raquelle I.; Metspalu, Andres; Michie, Patricia T.; Milani, Lili; Milanova, Vihra; Mokrab, Younes; Morris, Derek W.; Mors, Ole; Mortensen, Preben B.; Murphy, Kieran C.; Murray, Robin M.; Myin-Germeys, Inez; Mller-Myhsok, Bertram; Nelis, Mari; Nenadic, Igor; Nertney, Deborah A.; Nestadt, Gerald; Nicodemus, Kristin K.; Nikitina-Zake, Liene; Nisenbaum, Laura; Nordin, Annelie; O’Callaghan, Eadbhard; O’Dushlaine, Colm; O’Neill, F. Anthony; Oh, Sang-Yun; Olincy, Ann; Olsen, Line; Van Os, Jim; Pantelis, Christos; Papadimitriou, George N.; Papiol, Sergi; Parkhomenko, Elena; Pato, Michele T.; Paunio, Tiina; Pejovic-Milovancevic, Milica; Perkins, Diana O.; Pietilinen, Olli; Pimm, Jonathan; Pocklington, Andrew J.; Powell, John; Price, Alkes; Pulver, Ann E.; Purcell, Shaun M.; Quested, Digby; Rasmussen, Henrik B.; Reichenberg, Abraham; Reimers, Mark A.; Richards, Alexander L.; Roffman, Joshua L.; Roussos, Panos; Ruderfer, Douglas M.; Salomaa, Veikko; Sanders, Alan R.; Schall, Ulrich; Schubert, Christian R.; Schulze, Thomas G.; Schwab, Sibylle G.; Scolnick, Edward M.; Scott, Rodney J.; Seidman, Larry J.; Shi, Jianxin; Sigurdsson, Engilbert; Silagadze, Teimuraz; Silverman, Jeremy M.; Sim, Kang; Slominsky, Petr; Smoller, Jordan W.; So, Hon-Cheong; Spencer, Chris C.A.; Stahl, Eli A.; Stefansson, Hreinn; Steinberg, Stacy; Stogmann, Elisabeth; Straub, Richard E.; Strengman, Eric; Strohmaier, Jana; Stroup, T. Scott; Subramaniam, Mythily; Suvisaari, Jaana; Svrakic, Dragan M.; Szatkiewicz, Jin P.; Sderman, Erik; Thirumalai, Srinivas; Toncheva, Draga; Tooney, Paul A.; Tosato, Sarah; Veijola, Juha

    2014-01-01

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common diseases to partition the heritability explained by genotyped SNPs (hg2) across functional categories (while accounting for shared variance due to linkage disequilibrium). Extensive simulations showed that in contrast to current estimates from GWAS summary statistics, the variance-component approach partitions heritability accurately under a wide range of complex-disease architectures. Across the 11 diseases DNaseI hypersensitivity sites (DHSs) from 217 cell types spanned 16% of imputed SNPs (and 24% of genotyped SNPs) but explained an average of 79% (SE = 8%) of hg2 from imputed SNPs (5.1× enrichment; p = 3.7 × 10−17) and 38% (SE = 4%) of hg2 from genotyped SNPs (1.6× enrichment, p = 1.0 × 10−4). Further enrichment was observed at enhancer DHSs and cell-type-specific DHSs. In contrast, coding variants, which span 1% of the genome, explained <10% of hg2 despite having the highest enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease. PMID:25439723

  14. Neurophysiology of space travel: energetic solar particles cause cell type-specific plasticity of neurotransmission.

    Science.gov (United States)

    Lee, Sang-Hun; Dudok, Barna; Parihar, Vipan K; Jung, Kwang-Mook; Zöldi, Miklós; Kang, Young-Jin; Maroso, Mattia; Alexander, Allyson L; Nelson, Gregory A; Piomelli, Daniele; Katona, István; Limoli, Charles L; Soltesz, Ivan

    2016-11-30

    In the not too distant future, humankind will embark on one of its greatest adventures, the travel to distant planets. However, deep space travel is associated with an inevitable exposure to radiation fields. Space-relevant doses of protons elicit persistent disruptions in cognition and neuronal structure. However, whether space-relevant irradiation alters neurotransmission is unknown. Within the hippocampus, a brain region crucial for cognition, perisomatic inhibitory control of pyramidal cells (PCs) is supplied by two distinct cell types, the cannabinoid type 1 receptor (CB1)-expressing basket cells (CB1BCs) and parvalbumin (PV)-expressing interneurons (PVINs). Mice subjected to low-dose proton irradiation were analyzed using electrophysiological, biochemical and imaging techniques months after exposure. In irradiated mice, GABA release from CB1BCs onto PCs was dramatically increased. This effect was abolished by CB1 blockade, indicating that irradiation decreased CB1-dependent tonic inhibition of GABA release. These alterations in GABA release were accompanied by decreased levels of the major CB1 ligand 2-arachidonoylglycerol. In contrast, GABA release from PVINs was unchanged, and the excitatory connectivity from PCs to the interneurons also underwent cell type-specific alterations. These results demonstrate that energetic charged particles at space-relevant low doses elicit surprisingly selective long-term plasticity of synaptic microcircuits in the hippocampus. The magnitude and persistent nature of these alterations in synaptic function are consistent with the observed perturbations in cognitive performance after irradiation, while the high specificity of these changes indicates that it may be possible to develop targeted therapeutic interventions to decrease the risk of adverse events during interplanetary travel.

  15. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    Directory of Open Access Journals (Sweden)

    Stantchev Tzanko S

    2012-12-01

    Full Text Available Abstract Background The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI and/or thioredoxin (Trx have emerged as the two enzymes most often implicated in this process. Results We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid (DTNB, significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM, and also phytohemagglutinin (PHA-stimulated peripheral blood mononuclear cells (PBMC. Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb in HIV-1 envelope pseudotyped and wild type (wt virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Conclusions Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this

  16. Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.

    Science.gov (United States)

    Guzzo, Rosa M; Scanlon, Vanessa; Sanjay, Archana; Xu, Ren-He; Drissi, Hicham

    2014-12-01

    The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.

  17. Regulatory Domain Selectivity in the Cell-Type Specific PKN-Dependence of Cell Migration

    OpenAIRE

    Sylvie Lachmann; Amy Jevons; Manu De Rycker; Adele Casamassima; Simone Radtke; Alejandra Collazos; Peter J Parker

    2011-01-01

    The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relativel...

  18. Construction of cell type-specific logic models of signaling networks using CellNOpt.

    Science.gov (United States)

    Morris, Melody K; Melas, Ioannis; Saez-Rodriguez, Julio

    2013-01-01

    Mathematical models are useful tools for understanding protein signaling networks because they provide an integrated view of pharmacological and toxicological processes at the molecular level. Here we describe an approach previously introduced based on logic modeling to generate cell-specific, mechanistic and predictive models of signal transduction. Models are derived from a network encoding prior knowledge that is trained to signaling data, and can be either binary (based on Boolean logic) or quantitative (using a recently developed formalism, constrained fuzzy logic). The approach is implemented in the freely available tool CellNetOptimizer (CellNOpt). We explain the process CellNOpt uses to train a prior knowledge network to data and illustrate its application with a toy example as well as a realistic case describing signaling networks in the HepG2 liver cancer cell line.

  19. Cell-Type Specific Inactivation of Hippocampal CA1 Disrupts Location-Dependent Object Recognition in the Mouse

    Science.gov (United States)

    Haettig, Jakob; Sun, Yanjun; Wood, Marcelo A.; Xu, Xiangmin

    2013-01-01

    The allatostatin receptor (AlstR)/ligand inactivation system enables potent regulation of neuronal circuit activity. To examine how different cell types participate in memory formation, we have used this system through Cre-directed, cell-type specific expression in mouse hippocampal CA1 in vivo and examined functional effects of inactivation of…

  20. Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

    Science.gov (United States)

    Lachmann, Sylvie; Jevons, Amy; De Rycker, Manu; Casamassima, Adele; Radtke, Simone; Collazos, Alejandra; Parker, Peter J

    2011-01-01

    The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

  1. Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

    Directory of Open Access Journals (Sweden)

    Sylvie Lachmann

    Full Text Available The mammalian protein kinase N (PKN family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

  2. CAST-ChIP Maps Cell-Type-Specific Chromatin States in the Drosophila Central Nervous System

    Directory of Open Access Journals (Sweden)

    Tamás Schauer

    2013-10-01

    Full Text Available Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP, a broadly applicable biochemical procedure. RNA polymerase II (Pol II CAST-ChIP identifies ∼1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

  3. Advances in plant cell type-specific genome-wide studies of gene expression

    Institute of Scientific and Technical Information of China (English)

    Ying WANG; Yuling JIAO

    2011-01-01

    Cell is the functional unit of life.To study the complex interactions of systems of biological molecules,it is crucial to dissect these molecules at the cell level.In recent years,major progresses have been made by plant biologists to profile gene expression in specific cell types at the genome-wide level.Approaches based on the isolation of cells,polysomes or nuclei have been developed and successfully used for studying the cell types from distinct organs of several plant species.These cell-level data sets revealed previously unrecognized cellular properties,such as cell-specific gene expression modules and hormone response centers,and should serve as essential resources for functional genomic analyses.Newly developed technologies are more affordable to many laboratories and should help to provide new insights at the cellular resolution in the near future.

  4. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  5. Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

    Science.gov (United States)

    Coetzee, Simon G; Shen, Howard C; Hazelett, Dennis J; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J; Couch, Fergus J; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N A; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A; Pharoah, Paul D P; Noushmehr, Houtan; Gayther, Simon A

    2015-07-01

    Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer.

  6. Cell-type-specific neuroanatomy of cliques of autism-related genes in the mouse brain

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    Pascal eGrange

    2015-05-01

    Full Text Available Two cliques of genes identified computationally for their high co-expression in the mouse brain according to the Allen Brain Atlas, and for their enrichment in genes related to autism spectrum disorder, have recently been shown to be highly co-expressed in the cerebellar cortex, compared to what could be expected by chance. Moreover, the expression of these cliques of genes is not homogeneous across the cerebellar cortex, and it has been noted that their expression pattern seems to highlight the granular layer. However, this observation was only made by eye, and recent advances in computational neuroanatomy allow to rank cell types in the mouse brain (characterized by their transcriptome profiles according to the similarity between their spatial density profiles and the expression profiles of the cliques. We establish by Monte Carlo simulation that with probability at least 99%, the expression profiles of the two cliques are more similar to the density profile of granule cells than 99% of the expression of cliques containing the same number of genes (Purkinje cells also score above 99% in one of the cliques. Thresholding the expression profiles shows that the signal is more intense in the granular layer. Finally, we work out pairs of cell types whose combined expression profiles are more similar to the expression profiles of the cliquesthan any single cell type. These pairs predominantly consist of one cortical pyramidal cell and one cerebellar cell (whichcan be either a granule cell or a Purkinje cell.

  7. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity

    KAUST Repository

    Noutsi, Pakiza

    2016-06-30

    Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  8. Assessment of Membrane Fluidity Fluctuations during Cellular Development Reveals Time and Cell Type Specificity.

    Directory of Open Access Journals (Sweden)

    Pakiza Noutsi

    Full Text Available Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  9. Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers.

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    2006-01-01

    Full Text Available BACKGROUND: Inadequate oxygen (hypoxia triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF, plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases. METHODS AND FINDINGS: We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use. CONCLUSIONS: The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis

  10. Mouse Testicular Cell Type-Specific Antiviral Response against Mumps Virus Replication

    Science.gov (United States)

    Wu, Han; Zhao, Xiang; Wang, Fei; Jiang, Qian; Shi, Lili; Gong, Maolei; Liu, Weihua; Gao, Bo; Song, Chengyi; Li, Qihan; Chen, Yongmei; Han, Daishu

    2017-01-01

    Mumps virus (MuV) infection has high tropism to the testis and usually leads to orchitis, an etiological factor in male infertility. However, MuV replication in testicular cells and the cellular antiviral responses against MuV are not fully understood. The present study showed that MuV infected the majority of testicular cells, including Leydig cells (LC), testicular macrophages, Sertoli cells (SC), and male germ cells (GC). MuV was replicated at relatively high efficiencies in SC compared with LC and testicular macrophages. In contrast, MuV did not replicate in male GC. Notably, testicular cells exhibited different innate antiviral responses against MuV replication. We showed that interferon β (IFN-β) inhibited MuV replication in LC, macrophages, and SC, which were associated with the upregulation of major antiviral proteins. We provided primary evidence that autophagy plays a role in blocking MuV replication in male GC. Autophagy was also involved in limiting MuV replication in testicular macrophages but not in Leydig and SC. These findings indicate the involvement of the innate defense against MuV replication in testicular cells. PMID:28239382

  11. Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

    Science.gov (United States)

    Woclawek-Potocka, Izabela; Acosta, Tomas J; Korzekwa, Anna; Bah, Mamadou M; Shibaya, Masami; Okuda, Kiyoshi; Skarzynski, Dariusz J

    2005-05-01

    Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine

  12. Cell type-specific tuning of hippocampal interneuron firing during gamma oscillations in vivo.

    Science.gov (United States)

    Tukker, John J; Fuentealba, Pablo; Hartwich, Katja; Somogyi, Peter; Klausberger, Thomas

    2007-08-01

    Cortical gamma oscillations contribute to cognitive processing and are thought to be supported by perisomatic-innervating GABAergic interneurons. We performed extracellular recordings of identified interneurons in the hippocampal CA1 area of anesthetized rats, revealing that the firing patterns of five distinct interneuron types are differentially correlated to spontaneous gamma oscillations. The firing of bistratified cells, which target dendrites of pyramidal cells coaligned with the glutamatergic input from hippocampal area CA3, is strongly phase locked to field gamma oscillations. Parvalbumin-expressing basket, axo-axonic, and cholecystokinin-expressing interneurons exhibit moderate gamma modulation, whereas the spike timing of distal dendrite-innervating oriens-lacunosum moleculare interneurons is not correlated to field gamma oscillations. Cholecystokinin-expressing interneurons fire earliest in the gamma cycle, a finding that is consistent with their suggested function of thresholding individual pyramidal cells. Furthermore, we show that field gamma amplitude correlates with interneuronal spike-timing precision and firing rate. Overall, our recordings suggest that gamma synchronization in vivo is assisted by temporal- and domain-specific GABAergic inputs to pyramidal cells and is initiated in pyramidal cell dendrites in addition to somata and axon initial segments.

  13. Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

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    Cyrill Géraud

    Full Text Available Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ, i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

  14. Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain.

    Science.gov (United States)

    Shelbourne, Peggy F; Keller-McGandy, Christine; Bi, Wenya Linda; Yoon, Song-Ro; Dubeau, Louis; Veitch, Nicola J; Vonsattel, Jean Paul; Wexler, Nancy S; Arnheim, Norman; Augood, Sarah J

    2007-05-15

    Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

  15. Highly efficient cell-type-specific gene inactivation reveals a key function for the Drosophila FUS homolog cabeza in neurons.

    Science.gov (United States)

    Frickenhaus, Marie; Wagner, Marina; Mallik, Moushami; Catinozzi, Marica; Storkebaum, Erik

    2015-03-16

    To expand the rich genetic toolkit of Drosophila melanogaster, we evaluated whether introducing FRT or LoxP sites in endogenous genes could allow for cell-type-specific gene inactivation in both dividing and postmitotic cells by GAL4-driven expression of FLP or Cre recombinase. For proof of principle, conditional alleles were generated for cabeza (caz), the Drosophila homolog of human FUS, a gene implicated in the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Upon selective expression in neurons or muscle, both FLP and Cre mediated caz inactivation in all neurons or muscle cells, respectively. Neuron-selective caz inactivation resulted in failure of pharate adult flies to eclose from the pupal case, and adult escapers displayed motor performance defects and reduced life span. Due to Cre-toxicity, FLP/FRT is the preferred system for cell-type-specific gene inactivation, and this strategy outperforms RNAi-mediated knock-down. Furthermore, the GAL80 target system allowed for temporal control over gene inactivation, as induction of FLP expression from the adult stage onwards still inactivated caz in >99% of neurons. Remarkably, selective caz inactivation in adult neurons did not affect motor performance and life span, indicating that neuronal caz is required during development, but not for maintenance of adult neuronal function.

  16. Cardiovascular protection of magnolol: cell-type specificity and dose-related effects

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    Ho Jennifer

    2012-07-01

    Full Text Available Abstract Magnolia officinalis has been widely used in traditional Chinese medicine. Magnolol, an active component isolated from Magnolia officinalis, is known to be a cardiovascular protector since 1994. The multiplex mechanisms of magnolol on cardiovascular protection depends on cell types and dosages, and will be reviewed and discussed in this article. Magnolol under low and moderate dosage possesses the ability to protect heart from ischemic/reperfusion injury, reduces atherosclerotic change, protects endothelial cell against apoptosis and inhibits neutrophil-endothelial adhesion. The moderate to high concentration of magnolol mainly acts on smooth muscle cells and platelets. Magnolol induces apoptosis in vascular smooth muscle cells at moderate concentration and inhibits proliferation at moderate and high concentration. High concentration of magnolol also abrogates platelet activation, aggregation and thrombus formation. Magnolol also serves as an smooth muscle relaxant only upon the high concentration. Oral intake of magnolol to reach the therapeutic level for cardiovascular protection is applicable, thus makes magnolol an agent of great potential for preventing cardiovascular diseases in high-risk patients.

  17. Dopaminergic neurons write and update memories with cell-type-specific rules.

    Science.gov (United States)

    Aso, Yoshinori; Rubin, Gerald M

    2016-07-21

    Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. Previously we described the anatomy of the adult MB and defined 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments (Aso et al., 2014a, 2014b). Here we compare the properties of memories formed by optogenetic activation of individual DAN cell types. We found extensive differences in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. Our results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences.

  18. Innate immune response to pulmonary contusion: Identification of cell-type specific inflammatory responses

    OpenAIRE

    Hoth, J. Jason; Wells, Jonathan D.; Yoza, Barbara K.; McCall, Charles E.

    2012-01-01

    Lung injury from pulmonary contusion is a common traumatic injury, predominantly seen after blunt chest trauma such as in vehicular accidents. The local and systemic inflammatory response to injury includes activation of innate immune receptors, elaboration of a variety inflammatory mediators, and recruitment of inflammatory cells to the injured lung. Using a mouse model of pulmonary contusion, we had previously shown that innate immune Toll like receptors 2 and 4 (TLR2 and TLR4) mediate the ...

  19. One-pot synthesis of aptamer-functionalized silver nanoclusters for cell-type-specific imaging.

    Science.gov (United States)

    Li, Jingjing; Zhong, Xiaoqin; Cheng, Fangfang; Zhang, Jian-Rong; Jiang, Li-Ping; Zhu, Jun-Jie

    2012-05-01

    As an emerging category of fluorescent metal nanoclusters, oligonucleotide-templated silver nanoclusters (Ag NCs) have attracted a lot of interest and have shown wide application in biorelated disciplines. However, the weak fluorescence emission and poor permeability to cell membranes tethered further intracellular applications of Ag NCs. AS1411 is an antiproliferative G-rich phosphodiester oligonucleotide and currently an anticancer agent under phase II clinical trials. Herein, we present a strategy to synthesize AS1411-functionalized Ag NCs with excellent fluorescence through a facile one-pot process. Confocal laser scanning microscopy and Z-axis scanning confirmed that the AS1411-functionalized Ag NCs could be internalized into MCF-7 human breast cancer cells and were able to specifically stain nuclei with red color. To our surprise, 3-[4,5-dimethylthiazol-z-yl]-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated the Ag NCs were cytocompatible and showed better inhibition effects than pure AS1411 on MCF-7 human breast cancer cells. In addition, a universal design of the oligonucleotide scaffold for synthesis of Ag NCs was extended to other aptamers, such as Sgc8c and mucin 1 aptamer. Due to the facile synthesis procedure and capability of specific target recognition, this fluorescent platform will potentially broaden the applications of Ag NCs in biosensing and biological imaging.

  20. Cell-Type-Specific Activity in Prefrontal Cortex during Goal-Directed Behavior.

    Science.gov (United States)

    Pinto, Lucas; Dan, Yang

    2015-07-15

    The prefrontal cortex (PFC) plays a key role in controlling goal-directed behavior. Although a variety of task-related signals have been observed in the PFC, whether they are differentially encoded by various cell types remains unclear. Here we performed cellular-resolution microendoscopic Ca(2+) imaging from genetically defined cell types in the dorsomedial PFC of mice performing a PFC-dependent sensory discrimination task. We found that inhibitory interneurons of the same subtype were similar to each other, but different subtypes preferentially signaled different task-related events: somatostatin-positive neurons primarily signaled motor action (licking), vasoactive intestinal peptide-positive neurons responded strongly to action outcomes, whereas parvalbumin-positive neurons were less selective, responding to sensory cues, motor action, and trial outcomes. Compared to each interneuron subtype, pyramidal neurons showed much greater functional heterogeneity, and their responses varied across cortical layers. Such cell-type and laminar differences in neuronal functional properties may be crucial for local computation within the PFC microcircuit.

  1. Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes

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    Mathieu F. M. Cellier

    2013-01-01

    Full Text Available The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1 transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods. SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS, including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases.

  2. Cell type specificity and mechanism of control of a gene may be reverted in different strains of Dictyostelium discoideum.

    Science.gov (United States)

    Mangiarotti, G; Giorda, R

    2000-06-21

    Twelve genes which are expressed exclusively in pre-spore cells of Dictyostelium strain AX3 are expressed exclusively in pre-stalk cells of strain AX2. One gene has the opposite behavior: it is expressed in pre-stalk cells in AX3 and in pre-spore cells in AX2. The change in cell type specificity involves a change in the mechanism of control of gene expression. When they are expressed in pre-stalk cells, genes are controlled at the level of transcription, whilst in pre-spore cells, they are controlled at the level of mRNA stability. Genes expressed in pre-stalk cells in strain AX2, fused with an AX2 pre-spore specific promoter, become regulated at the level of mRNA stability. These findings indicate that at least a group of pre-stalk mRNAs possess the cis-destabilizing element typical of pre-spore mRNAs, though they are not destabilized in disaggregated cells. This is due to the fact that ribosomal protein S6, phosphorylation of which is responsible for controlling the stability of pre-spore mRNAs, is not dephosphorylated in disaggregated pre-stalk cells. These cells lack an S6 phosphatase activity which has been purified from disaggregated pre-spore cells.

  3. Cell-type specific mechanisms of D-serine uptake and release in the brain

    Directory of Open Access Journals (Sweden)

    Magalie eMartineau

    2014-05-01

    Full Text Available Accumulating evidence during the last decade established that D-serine is a key signaling molecule utilized by neurons and astroglia in the mammalian central nervous system. D-serine is increasingly appreciated as the main physiological endogenous coagonist for synaptic NMDA receptors at central excitatory synapses; it is mandatory for long-term changes in synaptic strength, memory, learning, and social interactions. Alterations in the extracellular levels of D-serine leading to disrupted cell-cell signaling are a trademark of many chronic or acute neurological (i.e. Alzheimer disease, epilepsy, stroke and psychiatric (i.e. schizophrenia disorders, and are associated with addictive behavior (i.e. cocaine addiction. Indeed, fine tuning of the extracellular levels of D-serine, achieved by various molecular machineries and signaling pathways, is necessary for maintenance of accurate NMDA receptor functions. Here, we review the experimental data supporting the notion that astroglia and neurons use different pathways to regulate levels of extracellular D-serine.

  4. Cell-Type-Specific Sensorimotor Processing in Striatal Projection Neurons during Goal-Directed Behavior.

    Science.gov (United States)

    Sippy, Tanya; Lapray, Damien; Crochet, Sylvain; Petersen, Carl C H

    2015-10-21

    Goal-directed sensorimotor transformation drives important aspects of mammalian behavior. The striatum is thought to play a key role in reward-based learning and action selection, receiving glutamatergic sensorimotor signals and dopaminergic reward signals. Here, we obtain whole-cell membrane potential recordings from the dorsolateral striatum of mice trained to lick a reward spout after a whisker deflection. Striatal projection neurons showed strong task-related modulation, with more depolarization and action potential firing on hit trials compared to misses. Direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, exhibited a prominent early sensory response. Optogenetic stimulation of direct pathway striatonigral neurons, but not indirect pathway striatopallidal neurons, readily substituted for whisker stimulation evoking a licking response. Our data are consistent with direct pathway striatonigral neurons contributing a "go" signal for goal-directed sensorimotor transformation leading to action initiation. VIDEO ABSTRACT.

  5. Investigation of Neuronal Cell Type-Specific Gene Expression of Ca2+/Calmodulin-dependent Protein Kinase II.

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    Mima Kazuko

    2002-01-01

    Full Text Available The promoter activity of the rat Ca2+/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-flanking region of &agr; and &bgr; isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5'-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types.

  6. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

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    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  7. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    Science.gov (United States)

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

  8. Cell type-specific thalamic innervation in a column of rat vibrissal cortex.

    Science.gov (United States)

    Meyer, Hanno S; Wimmer, Verena C; Hemberger, Mike; Bruno, Randy M; de Kock, Christiaan P J; Frick, Andreas; Sakmann, Bert; Helmstaedter, Moritz

    2010-10-01

    This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

  9. Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit.

    Science.gov (United States)

    Vickers, J C; Morrison, J H; Friedrich, V L; Elder, G A; Perl, D P; Katz, R N; Lazzarini, R A

    1994-09-01

    Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2-25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

  10. Human brain derived cells respond in a type-specific manner after exposure to urban particulate matter (PM).

    Science.gov (United States)

    Campbell, Arezoo; Daher, Nancy; Solaimani, Parrisa; Mendoza, Kriscelle; Sioutas, Constantinos

    2014-10-01

    Exposure to particulate matter (PM), a component of urban air pollution, may cause adverse effects in the brain. Although the exact mechanisms involved are unknown, both oxidative and inflammatory responses have been reported. Since the main route of exposure to particulate matter is through inhalation, there is a potential for compounds to directly enter the brain and alter normal cellular function. Enhancement in both oxidative stress and neuroinflammatory markers has been observed in neurodegenerative disorders and PM-induced potentiation of these events may accelerate the disease process. The objective of this pilot study was to use normal human brain cells, a model system which has not been previously used, to assess cell-type-specific responses after exposure to ultrafine particles (UFP). Human microglia, neurons, and astrocytes were grown separately or as co-cultures and then exposed to aqueous UFP suspensions. Reactive Oxygen Species (ROS) formation and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) were measured as markers of oxidative stress or inflammation respectively. Our results revealed that after exposure to 2 μg/ml of particles, normal human neurons exhibit a decrease in ROS formation and an increase in TNF-α. The observed decrease in ROS formation persisted in the presence of glial cells, which contrasts previous studies done in rodent cells reporting that PM-induced microglial activation modulates neuronal responses. Our study indicates that human CNS cells may respond differently compared to rodent cells and that their use may be more predictive in risk assessment.

  11. Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

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    Anja eScharinger

    2015-08-01

    Full Text Available Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM. It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA. Using these mice we provide biochemical evidence for the existence of long (CTM-containing and short (CTM-deficient Cav1.3 α1-subunits in brain. The long (HA-labeled Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It is required to stabilize gating properties of Cav1.3 channels required for normal electrical excitability.

  12. Cell-type-specific recruitment of amygdala interneurons to hippocampal theta rhythm and noxious stimuli in vivo.

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    Bienvenu, Thomas C M; Busti, Daniela; Magill, Peter J; Ferraguti, Francesco; Capogna, Marco

    2012-06-21

    Neuronal synchrony in the basolateral amygdala (BLA) is critical for emotional behavior. Coordinated theta-frequency oscillations between the BLA and the hippocampus and precisely timed integration of salient sensory stimuli in the BLA are involved in fear conditioning. We characterized GABAergic interneuron types of the BLA and determined their contribution to shaping these network activities. Using in vivo recordings in rats combined with the anatomical identification of neurons, we found that the firing of BLA interneurons associated with network activities was cell type specific. The firing of calbindin-positive interneurons targeting dendrites was precisely theta-modulated, but other cell types were heterogeneously modulated, including parvalbumin-positive basket cells. Salient sensory stimuli selectively triggered axo-axonic cells firing and inhibited firing of a disctinct projecting interneuron type. Thus, GABA is released onto BLA principal neurons in a time-, domain-, and sensory-specific manner. These specific synaptic actions likely cooperate to promote amygdalo-hippocampal synchrony involved in emotional memory formation.

  13. Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

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    Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.; Lee, Mihwa; Craig, Vanessa J.; Bach, Ingolf; Guss, J. Mitchell; Mackay, Joel P.; Matthews, Jacqueline M. (UMASS, MED); (Sydney)

    2008-09-03

    LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}). Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.

  14. Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features.

    Science.gov (United States)

    Yáñez-Cuna, J Omar; Arnold, Cosmas D; Stampfel, Gerald; Boryń, Lukasz M; Gerlach, Daniel; Rath, Martina; Stark, Alexander

    2014-07-01

    Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

  15. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

    Science.gov (United States)

    Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

    2015-06-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  16. Cell-type-specific expression of STAT transcription factors in tissue samples from patients with lymphocytic thyroiditis.

    Science.gov (United States)

    Staab, Julia; Barth, Peter J; Meyer, Thomas

    2012-09-01

    Expression of cytokine-regulated signal transducer and activator of transcription (STAT) proteins was histochemically assessed in patients diagnosed as having Hashimoto's disease or focal lymphocytic thyroiditis (n = 10). All surgical specimens showed histological features of lymphocytic thyroiditis, including a diffuse infiltration with mononuclear cells and an incomplete loss of thyroid follicles, resulting in the destruction of glandular tissue architecture. Immunohistochemical analysis demonstrated differential expression patterns of the various members of the STAT transcription factors examined, indicating that each member of this conserved protein family has its distinct functions in the development of the disease. Using an antibody that specifically recognized the phosphorylated tyrosine residue in position 701, we detected activated STAT1 dimers in numerous germinal macrophages and infiltrating lymphocytes as well as in oncocytes. In contrast, STAT3 expression was restricted to epithelial cells and showed a clear colocalization with the antiapoptotic protein Bcl-2. Moreover, expression of phospho-STAT3 was associated with low levels of stromal fibrosis, suggesting that STAT3 serves as a protective factor in the remodeling of the inflamed thyroid gland. Phospho-STAT5 immunoreactivity was detected in numerous infiltrating cells of hematopoietic origin and, additionally, in hyperplastic follicular epithelia. This tissue distribution demonstrated that activated STAT5 molecules participate in both lymphocytopoiesis and possibly also in the buildup of regenerating thyroid follicles. Taken together, the cell-type-specific expression patterns of STAT proteins in human lymphocytic thyroiditis reflect their distinct and partially antagonistic roles in orchestrating the balance between degenerating and regenerating processes within a changing cytokine environment.

  17. Adolescent maturation of inhibitory inputs onto cingulate cortex neurons is cell-type specific and TrkB dependent

    Directory of Open Access Journals (Sweden)

    Angela eVandenberg

    2015-02-01

    Full Text Available The maturation of inhibitory circuits during adolescence may be tied to the onset of mental health disorders such as schizophrenia. Neurotrophin signaling likely plays a critical role in supporting inhibitory circuit development and is also implicated in psychiatric disease. Within the neocortex, subcircuits may mature at different times and show differential sensitivity to neurotrophin signaling. We measured miniature inhibitory and excitatory postsynaptic currents (mIPSC and mEPSCs in Layer 5 cell-types in the mouse anterior cingulate across the periadolescent period. We differentiated cell-types mainly by Thy1 YFP transgene expression and also retrobead injection labeling in the contralateral cingulate and ipsilateral pons. We found that YFP- neurons and commissural projecting neurons had lower frequency of mIPSCs than neighboring YFP+ neurons or pons projecting neurons in juvenile mice (P21-25. YFP- neurons and to a lesser extent commissural projecting neurons also showed a significant increase in mIPSC amplitude during the periadolescent period (P21-25 vs. P40-50, which was not seen in YFP+ neurons or pons projecting neurons. Systemic disruption of tyrosine kinase receptor B (TrkB signaling during P23-50 in TrkBF616A mice blocked developmental changes in mIPSC amplitude, without affecting miniature excitatory post synaptic currents (mEPSCs. Our data suggest that the maturation of inhibitory inputs onto layer 5 pyramidal neurons is cell-type specific. These data may inform our understanding of adolescent brain development across species and aid in identifying candidate subcircuits that may show greater vulnerability in mental illness.

  18. Post-ischaemic long-term synaptic potentiation in the striatum: a putative mechanism for cell type-specific vulnerability.

    Science.gov (United States)

    Calabresi, Paolo; Saulle, Emilia; Centonze, Diego; Pisani, Antonio; Marfia, Girolama A; Bernardi, Giorgio

    2002-04-01

    In the present in vitro study of rat brain, we report that transient oxygen and glucose deprivation (in vitro ischaemia) induced a post-ischaemic long-term synaptic potentiation (i-LTP) at corticostriatal synapses. We compared the physiological and pharmacological characteristics of this pathological form of synaptic plasticity with those of LTP induced by tetanic stimulation of corticostriatal fibres (t-LTP), which is thought to represent a cellular substrate of learning and memory. Activation of N-methyl-D-aspartate (NMDA) receptors was required for the induction of both forms of synaptic plasticity. The intraneuronal injection of the calcium chelator BAPTA [bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate] and inhibitors of the mitogen-activated protein kinase pathway blocked both forms of synaptic plasticity. However, while t-LTP showed input specificity, i-LTP occurred also at synaptic pathways inactive during the ischaemic period. In addition, scopolamine, a muscarinic receptor antagonist, prevented the induction of t-LTP but not of i-LTP, indicating that endogenous acetylcholine is required for physiological but not for pathological synaptic potentiation. Finally, we found that striatal cholinergic interneurones, which are resistant to in vivo ischaemia, do not express i-LTP while they express t-LTP. We suggest that i-LTP represents a pathological form of synaptic plasticity that may account for the cell type-specific vulnerability observed in striatal spiny neurones following ischaemia and energy deprivation.

  19. Cell type-specific genes show striking and distinct patterns of spatial expression in the mouse brain.

    Science.gov (United States)

    Ko, Younhee; Ament, Seth A; Eddy, James A; Caballero, Juan; Earls, John C; Hood, Leroy; Price, Nathan D

    2013-02-19

    To characterize gene expression patterns in the regional subdivisions of the mammalian brain, we integrated spatial gene expression patterns from the Allen Brain Atlas for the adult mouse with panels of cell type-specific genes for neurons, astrocytes, and oligodendrocytes from previously published transcriptome profiling experiments. We found that the combined spatial expression patterns of 170 neuron-specific transcripts revealed strikingly clear and symmetrical signatures for most of the brain's major subdivisions. Moreover, the brain expression spatial signatures correspond to anatomical structures and may even reflect developmental ontogeny. Spatial expression profiles of astrocyte- and oligodendrocyte-specific genes also revealed regional differences; these defined fewer regions and were less distinct but still symmetrical in the coronal plane. Follow-up analysis suggested that region-based clustering of neuron-specific genes was related to (i) a combination of individual genes with restricted expression patterns, (ii) region-specific differences in the relative expression of functional groups of genes, and (iii) regional differences in neuronal density. Products from some of these neuron-specific genes are present in peripheral blood, raising the possibility that they could reflect the activities of disease- or injury-perturbed networks and collectively function as biomarkers for clinical disease diagnostics.

  20. DETECTION OF E6, E7 AND CELL-TYPE SPECIFIC ENHANCER OF HUMAN PAPILLOMAVIRUS TYPE 16 IN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    HE Qian; CHU Yong-lie; JIA Xiao-li; ZHANG Shu-qun; LIU Wen-kang

    2008-01-01

    Objective To detect HPV16 E6, E7 genes and cell-type specific enhancer (CTSE) of long control region (LCR) in breast carcinoma (BC).Methods HPV16 E6,E7 genes and CTSE were detected in 40 BCs and 20 normal breast tissue (NBT) using polymerase chain reaction (PCR).Results The positive rates of HPV16 E6, E7genes and CTSE were 60% (24/40),55% (22/40) and 67.5%(27/40)respectively in BCs, whereas only 5% (1/20), 5%(1/20) and 15% (3/20) in NBTs (P<0.05). There exited significant correlation between E6 gene and CTSE in BCs (P<0.05), as well as E7 gene and CTSE. The infection of HPV16 E6, E7 and CTSE had no statistic relationship with pathological features.Conclusion There were HPV16 E6, E7 genes and CTSE together in BCs and CTSE may play an important role in pathogenesis of BC.

  1. Fiber Type-Specific Satellite Cell Content in Cyclists Following Heavy Training with Carbohydrate and Carbohydrate-Protein Supplementation

    Science.gov (United States)

    McKenzie, Alec I.; D'Lugos, Andrew C.; Saunders, Michael J.; Gworek, Keith D.; Luden, Nicholas D.

    2016-01-01

    The central purpose of this study was to evaluate the fiber type-specific satellite cell and myonuclear responses of endurance-trained cyclists to a block of intensified training, when supplementing with carbohydrate (CHO) vs. carbohydrate-protein (PRO). In a crossover design, endurance-trained cyclists (n = 8) performed two consecutive training periods, once supplementing with CHO (de facto “control” condition) and the other with PRO. Each training period consisted of 10 days of intensified cycle training (ICT–120% increase in average training duration) followed by 10 days of recovery (RVT–reduced volume training; 33% volume reduction vs. normal training). Skeletal muscle biopsies were obtained from the vastus lateralis before and after ICT and again following RVT. Immunofluorescent microscopy was used to quantify SCs (Pax7+), myonuclei (DAPI+), and myosin heavy chain I (MyHC I). Data are expressed as percent change ± 90% confidence limits. The 10-day block of ICTCHO increased MyHC I SC content (35 ± 28%) and myonuclear density (16 ± 6%), which remained elevated following RVTCHO (SC = 69 ± 50% vs. PRE; Nuclei = 17 ± 15% vs. PRE). MyHC II SC and myonuclei were not different following ICTCHO, but were higher following RVTCHO (SC = +33 ± 31% vs. PRE; Nuclei = 15 ± 14% vs. PRE), indicating a delayed response compared to MyHC I fibers. The MyHC I SC pool increased following ICTPRO (37 ± 37%), but without a concomitant increase in myonuclei. There were no changes in MyHC II SC or myonuclei following ICTPRO. Collectively, these trained endurance cyclists possessed a relatively large pool of SCs that facilitated rapid (MyHC I) and delayed (MyHC II) satellite cell proliferation and myonuclear accretion under carbohydrate conditions. The current findings strengthen the growing body of evidence demonstrating alterations in satellite cell number in the absence of hypertrophy. Satellite cell pool expansion is typically viewed as an advantageous response to

  2. Cell-Type Specific Channelopathies in the Prefrontal Cortex of the fmr1-/y Mouse Model of Fragile X Syndrome.

    Science.gov (United States)

    Kalmbach, Brian E; Johnston, Daniel; Brager, Darrin H

    2015-01-01

    Fragile X syndrome (FXS) is caused by transcriptional silencing of the fmr1 gene resulting in the loss of fragile X mental retardation protein (FMRP) expression. FXS patients display several behavioral phenotypes associated with prefrontal cortex (PFC) dysfunction. Voltage-gated ion channels, some of which are regulated by FMRP, heavily influence PFC neuron function. Although there is evidence for brain region-specific alterations to the function a single type of ion channel in FXS, it is unclear whether subtypes of principal neurons within a brain region are affected uniformly. We tested for alterations to ion channels critical in regulating neural excitability in two subtypes of prefrontal L5 pyramidal neurons. Using somatic and dendritic patch-clamp recordings, we provide evidence that the functional expression of h-channels (Ih) is down-regulated, whereas A-type K(+) channel function is up-regulated in pyramidal tract-projecting (PT) neurons in the fmr1-/y mouse PFC. This is the opposite pattern of results from published findings from hippocampus where Ih is up-regulated and A-type K(+) channel function is down-regulated. Additionally, we find that somatic Kv1-mediated current is down-regulated, resulting in increased excitability of fmr1-/y PT neurons. Importantly, these h- and K(+) channel differences do not extend to neighboring intratelencephalic-projecting neurons. Thus, the absence of FMRP has divergent effects on the function of individual types of ion channels not only between brain regions, but also variable effects across cell types within the same brain region. Given the importance of ion channels in regulating neural circuits, these results suggest cell-type-specific phenotypes for the disease.

  3. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    Science.gov (United States)

    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties.

  4. Drug and cell type-specific regulation of genes with different classes of estrogen receptor beta-selective agonists.

    Directory of Open Access Journals (Sweden)

    Sreenivasan Paruthiyil

    Full Text Available Estrogens produce biological effects by interacting with two estrogen receptors, ERalpha and ERbeta. Drugs that selectively target ERalpha or ERbeta might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERbeta-selective compounds have been identified. One class of ERbeta-selective agonists is represented by ERB-041 (WAY-202041 which binds to ERbeta much greater than ERalpha. A second class of ERbeta-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERbeta. Diarylpropionitrile represents a third class of ERbeta-selective compounds because its selectivity is due to a combination of greater binding to ERbeta and transcriptional activity. However, it is unclear if these three classes of ERbeta-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERbeta selectivity and pattern of gene expression of these three classes of ERbeta-selective compounds compared to estradiol (E(2, which is a non-selective ER agonist. U2OS cells stably transfected with ERalpha or ERbeta were treated with E(2 or the ERbeta-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERbeta-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERbeta, which is consistent with the finding that most genes regulated by the ERbeta-selective compounds were similar to each other and E(2. However, there were some classes of genes differentially regulated by the ERbeta agonists and E(2. Two ERbeta-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERbeta. Our gene profiling studies

  5. Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs.

    Science.gov (United States)

    Wile, Brian M; Ban, Kiwon; Yoon, Young-Sup; Bao, Gang

    2014-10-01

    Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

  6. An atlas for Schistosoma mansoni organs and life-cycle stages using cell type-specific markers and confocal microscopy.

    Directory of Open Access Journals (Sweden)

    James J Collins

    Full Text Available Schistosomiasis (bilharzia is a tropical disease caused by trematode parasites (Schistosoma that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites.

  7. Coordinated cell type-specific epigenetic remodeling in prefrontal cortex begins before birth and continues into early adulthood.

    Directory of Open Access Journals (Sweden)

    Hennady P Shulha

    2013-04-01

    Full Text Available Development of prefrontal and other higher-order association cortices is associated with widespread changes in the cortical transcriptome, particularly during the transitions from prenatal to postnatal development, and from early infancy to later stages of childhood and early adulthood. However, the timing and longitudinal trajectories of neuronal gene expression programs during these periods remain unclear in part because of confounding effects of concomitantly occurring shifts in neuron-to-glia ratios. Here, we used cell type-specific chromatin sorting techniques for genome-wide profiling of a histone mark associated with transcriptional regulation--H3 with trimethylated lysine 4 (H3K4me3--in neuronal chromatin from 31 subjects from the late gestational period to 80 years of age. H3K4me3 landscapes of prefrontal neurons were developmentally regulated at 1,157 loci, including 768 loci that were proximal to transcription start sites. Multiple algorithms consistently revealed that the overwhelming majority and perhaps all of developmentally regulated H3K4me3 peaks were on a unidirectional trajectory defined by either rapid gain or loss of histone methylation during the late prenatal period and the first year after birth, followed by similar changes but with progressively slower kinetics during early and later childhood and only minimal changes later in life. Developmentally downregulated H3K4me3 peaks in prefrontal neurons were enriched for Paired box (Pax and multiple Signal Transducer and Activator of Transcription (STAT motifs, which are known to promote glial differentiation. In contrast, H3K4me3 peaks subject to a progressive increase in maturing prefrontal neurons were enriched for activating protein-1 (AP-1 recognition elements that are commonly associated with activity-dependent regulation of neuronal gene expression. We uncovered a developmental program governing the remodeling of neuronal histone methylation landscapes in the prefrontal

  8. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

    Science.gov (United States)

    Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

    2016-09-01

    This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

  9. Differential microRNA expression signatures and cell type-specific association with Taxol resistance in ovarian cancer cells.

    Science.gov (United States)

    Kim, Yong-Wan; Kim, Eun Young; Jeon, Doin; Liu, Juinn-Lin; Kim, Helena Suhyun; Choi, Jin Woo; Ahn, Woong Shick

    2014-01-01

    Paclitaxel (Taxol) resistance remains a major obstacle for the successful treatment of ovarian cancer. MicroRNAs (miRNAs) have oncogenic and tumor suppressor activity and are associated with poor prognosis phenotypes. miRNA screenings for this drug resistance are needed to estimate the prognosis of the disease and find better drug targets. miRNAs that were differentially expressed in Taxol-resistant ovarian cancer cells, compared with Taxol-sensitive cells, were screened by Illumina Human MicroRNA Expression BeadChips. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to identify target genes of selected miRNAs. Kaplan-Meier survival analysis was applied to identify dysregulated miRNAs in ovarian cancer patients using data from The Cancer Genome Atlas. A total of 82 miRNAs were identified in ovarian carcinoma cells compared to normal ovarian cells. miR-141, miR-106a, miR-200c, miR-96, and miR-378 were overexpressed, and miR-411, miR-432, miR-494, miR-409-3p, and miR-655 were underexpressed in ovarian cancer cells. Seventeen miRNAs were overexpressed in Taxol-resistant cells, including miR-663, miR-622, and HS_188. Underexpressed miRNAs in Taxol-sensitive cells included miR-497, miR-187, miR-195, and miR-107. We further showed miR-663 and miR-622 as significant prognosis markers of the chemo-resistant patient group. In particular, the downregulation of the two miRNAs was associated with better survival, perhaps increasing the sensitivity of cancer cells to Taxol. In the chemo-sensitive patient group, only miR-647 could be a prognosis marker. These miRNAs inhibit several interacting genes of p53 networks, especially in TUOS-3 and TUOS-4, and showed cell line-specific inhibition effects. Taken together, the data indicate that the three miRNAs are closely associated with Taxol resistance and potentially better prognosis factors. Our results suggest that these miRNAs were successfully and reliably identified and would be used in the

  10. Differential microRNA expression signatures and cell type-specific association with Taxol resistance in ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Kim YW

    2014-02-01

    Full Text Available Yong-Wan Kim,1 Eun Young Kim,1 Doin Jeon,1 Juinn-Lin Liu,2 Helena Suhyun Kim,3 Jin Woo Choi,4 Woong Shick Ahn5 1Cancer Research Institute of Medical Science, The Catholic University of Korea, Seoul, Republic of Korea; 2Brain Tumor Center, Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, TX, USA; 3Cancer Rehab Laboratory, RH Healthcare Systems Inc, TX, USA; 4Harvard Medical School and Wellman Center for Photomedicine, Cambridge, MA, USA; 5Department of Obstetrics and Gynecology, The Catholic University of Korea, Seoul, Republic of Korea Abstract: Paclitaxel (Taxol resistance remains a major obstacle for the successful treatment of ovarian cancer. MicroRNAs (miRNAs have oncogenic and tumor suppressor activity and are associated with poor prognosis phenotypes. miRNA screenings for this drug resistance are needed to estimate the prognosis of the disease and find better drug targets. miRNAs that were differentially expressed in Taxol-resistant ovarian cancer cells, compared with Taxol-sensitive cells, were screened by Illumina Human MicroRNA Expression BeadChips. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR was used to identify target genes of selected miRNAs. Kaplan–Meier survival analysis was applied to identify dysregulated miRNAs in ovarian cancer patients using data from The Cancer Genome Atlas. A total of 82 miRNAs were identified in ovarian carcinoma cells compared to normal ovarian cells. miR-141, miR-106a, miR-200c, miR-96, and miR-378 were overexpressed, and miR-411, miR-432, miR-494, miR-409-3p, and miR-655 were underexpressed in ovarian cancer cells. Seventeen miRNAs were overexpressed in Taxol-resistant cells, including miR-663, miR-622, and HS_188. Underexpressed miRNAs in Taxol-sensitive cells included miR-497, miR-187, miR-195, and miR-107. We further showed miR-663 and miR-622 as significant prognosis markers of the chemo-resistant patient group. In particular, the

  11. Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells.

    Science.gov (United States)

    Braun, Doreen; Kinne, Anita; Bräuer, Anja U; Sapin, Remy; Klein, Marc O; Köhrle, Josef; Wirth, Eva K; Schweizer, Ulrich

    2011-03-01

    Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.

  12. Cell type-specific long-term plasticity at glutamatergic synapses onto hippocampal interneurons expressing either parvalbumin or CB1 cannabinoid receptor.

    Science.gov (United States)

    Nissen, Wiebke; Szabo, Andras; Somogyi, Jozsef; Somogyi, Peter; Lamsa, Karri P

    2010-01-27

    Different GABAergic interneuron types have specific roles in hippocampal function, and anatomical as well as physiological features vary greatly between interneuron classes. Long-term plasticity of interneurons has mostly been studied in unidentified GABAergic cells and is known to be very heterogeneous. Here we tested whether cell type-specific plasticity properties in distinct GABAergic interneuron types might underlie this heterogeneity. We show that long-term potentiation (LTP) and depression (LTD), two common forms of synaptic plasticity, are expressed in a highly cell type-specific manner at glutamatergic synapses onto hippocampal GABAergic neurons. Both LTP and LTD are generated in interneurons expressing parvalbumin (PV+), whereas interneurons with similar axon distributions but expressing cannabinoid receptor-1 show no lasting plasticity in response to the same protocol. In addition, LTP or LTD occurs in PV+ interneurons with different efferent target domains. Perisomatic-targeting PV+ basket and axo-axonic interneurons express LTP, whereas glutamatergic synapses onto PV+ bistratified cells display LTD. Both LTP and LTD are pathway specific, independent of NMDA receptors, and occur at synapses with calcium-permeable (CP) AMPA receptors. Plasticity in interneurons with CP-AMPA receptors strongly modulates disynaptic GABAergic transmission onto CA1 pyramidal cells. We propose that long-term plasticity adjusts the synaptic strength between pyramidal cells and interneurons in a cell type-specific manner and, in the defined CA1 interneurons, shifts the spatial pattern of inhibitory weight from pyramidal cell dendrites to the perisomatic region.

  13. Cell-Type-Specific Effects of Silibinin on Vitamin D-Induced Differentiation of Acute Myeloid Leukemia Cells Are Associated with Differential Modulation of RXRα Levels

    Directory of Open Access Journals (Sweden)

    Rina Wassermann

    2012-01-01

    Full Text Available Plant polyphenols have been shown to enhance the differentiation of acute myeloid leukemia (AML cells induced by the hormonal form of vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D. However, how these agents modulate 1,25D effects in different subtypes of AML cells remains poorly understood. Here, we show that both carnosic acid (CA and silibinin (SIL synergistically enhancd 1,25D-induced differentiation of myeloblastic HL60 cells. However, in promonocytic U937 cells, only CA caused potentiation while SIL attenuated 1,25D effect. The enhanced effect of 1,25D+CA was accompanied by increases in both the vitamin D receptor (VDR and retinoid X receptor alpha (RXRα protein levels and vitamin D response element (VDRE transactivation in both cell lines. Similar increases were observed in HL60 cells treated with 1,25D + SIL. In U937 cells, however, SIL inhibited 1,25D-induced VDRE transactivation concomitant with downregulation of RXRα at both transcriptional and posttranscriptional levels. These inhibitory effects correlated with the inability of SIL, with or without 1,25D, to activate the Nrf2/antioxidant response element signaling pathway in U937 cells. These results suggest that opposite effects of SIL on 1,25D-induced differentiation of HL60 and U937 cells may be determined by cell-type-specific signaling and transcriptional responses to this polyphenol resulting in differential modulation of RXRα expression.

  14. Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

    Science.gov (United States)

    Greiner, Alexandra M; Klein, Franziska; Gudzenko, Tetyana; Richter, Benjamin; Striebel, Thomas; Wundari, Bayu G; Autenrieth, Tatjana J; Wegener, Martin; Franz, Clemens M; Bastmeyer, Martin

    2015-11-01

    Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, β1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

  15. Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.

    Science.gov (United States)

    Diermeier-Daucher, Simone; Clarke, Scott T; Hill, Dani; Vollmann-Zwerenz, Arabel; Bradford, Jolene A; Brockhoff, Gero

    2009-06-01

    Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

  16. Microarray gene expression analysis to evaluate cell type specific expression of targets relevant for immunotherapy of hematological malignancies

    NARCIS (Netherlands)

    M.J. Pont (Margot); M.W. Honders; A.N. Kremer; C. van Kooten (Cees); C. Out; P.S. Hiemstra (Pieter); H.C. De Boer; M.J. Jager (Martine); E. Schmelzer; R.G.J. Vries (Robert); A.S. Al Hinai; W.G. Kroes (W.); R. Monajemi (Ramin); J.J. Goeman (Jelle); S. Böhringer (Stefan); W.A.F. Marijt; J.H.F. Falkenburg (Frederik); M. Griffioen

    2016-01-01

    textabstractCellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, de

  17. Folic acid induces cell type-specific changes in the transcriptome of breast cancer cell lines: a proof-of-concept study.

    Science.gov (United States)

    Price, R Jordan; Lillycrop, Karen A; Burdge, Graham C

    2016-01-01

    The effect of folic acid (FA) on breast cancer (BC) risk is uncertain. We hypothesised that this uncertainty may be due, in part, to differential effects of FA between BC cells with different phenotypes. To test this we investigated the effect of treatment with FA concentrations within the range of unmetabolised FA reported in humans on the expression of the transcriptome of non-transformed (MCF10A) and cancerous (MCF7 and Hs578T) BC cells. The total number of transcripts altered was: MCF10A, seventy-five (seventy up-regulated); MCF7, twenty-four (fourteen up-regulated); and Hs578T, 328 (156 up-regulated). Only the cancer-associated gene TAGLN was altered by FA in all three cell lines. In MCF10A and Hs578T cells, FA treatment decreased pathways associated with apoptosis, cell death and senescence, but increased those associated with cell proliferation. The folate transporters SLC19A1, SLC46A1 and FOLR1 were differentially expressed between cell lines tested. However, the level of expression was not altered by FA treatment. These findings suggest that physiological concentrations of FA can induce cell type-specific changes in gene regulation in a manner that is consistent with proliferative phenotype. This has implications for understanding the role of FA in BC risk. In addition, these findings support the suggestion that differences in gene expression induced by FA may involve differential activities of folate transporters. Together these findings indicate the need for further studies of the effect of FA on BC.

  18. The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

    Science.gov (United States)

    Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

    2001-05-01

    cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on

  19. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies

    OpenAIRE

    Pont, M. J.; Honders, M.W.; Kremer, A. N.; van Kooten, C.; C Out; Hiemstra, P. S.; de Boer, H. C.; Jager, M J; Schmelzer, E; Vries, R.G.; A S Al Hinai; Kroes, W. G.; Monajemi, R.; Goeman, J.J.; Böhringer, S

    2016-01-01

    Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimat...

  20. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Lojk J

    2015-02-01

    Full Text Available Jasna Lojk,1 Vladimir B Bregar,1 Maruša Rajh,1 Katarina Miš,2 Mateja Erdani Kreft,3 Sergej Pirkmajer,2 Peter Veranič,3 Mojca Pavlin1 1Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, 2Institute of Pathophysiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Abstract: Magnetic nanoparticles (NPs are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA in three cell types: Chinese Hamster Ovary (CHO, mouse melanoma (B16 cell line, and primary human myoblasts (MYO. We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better

  1. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    Directory of Open Access Journals (Sweden)

    M J Pont

    Full Text Available Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage-restricted expression as potential targets for immunotherapy of hematological cancers.

  2. Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

    Science.gov (United States)

    Pont, M J; Honders, M W; Kremer, A N; van Kooten, C; Out, C; Hiemstra, P S; de Boer, H C; Jager, M J; Schmelzer, E; Vries, R G; Al Hinai, A S; Kroes, W G; Monajemi, R; Goeman, J J; Böhringer, S; Marijt, W A F; Falkenburg, J H F; Griffioen, M

    2016-01-01

    Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

  3. Inflammatory cytokines IL-1β and TNF-α regulate p75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signalling mechanisms

    Directory of Open Access Journals (Sweden)

    Wilma J Friedman

    2009-05-01

    Full Text Available The p75NTR (where NTR is neurotrophin receptor can mediate many distinct cellular functions, including cell survival and apoptosis, axonal growth and cell proliferation, depending on the cellular context. This multifunctional receptor is widely expressed in the CNS (central nervous system during development, but its expression is restricted in the adult brain. However, p75NTR is induced by a variety of pathophysiological insults, including seizures, lesions and degenerative disease. We have demonstrated previously that p75NTR is induced by seizures in neurons, where it induces apoptosis, and in astrocytes, where it may regulate proliferation. In the present study, we have investigated whether the inflammatory cytokines IL (interleukin-1β and TNF-α (tumour necrosis factor-α, that are commonly elevated in these pathological conditions, mediate the regulation of p75NTR in neurons and astrocytes. We have further analysed the signal transduction pathways by which these cytokines induce p75NTR expression in the different cell types, specifically investigating the roles of the NF-κB (nuclear factor κB and p38 MAPK (mitogen-activated protein kinase pathways. We have demonstrated that both cytokines regulate p75NTR expression; however, the mechanisms governing this regulation are cytokine- and cell-type specific. The distinct mechanisms of cytokine-mediated p75NTR regulation that we demonstrate in the present study may facilitate therapeutic intervention in regulation of this receptor in a cell-selective manner.

  4. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data-Application to Monocyte Gene Regulation.

    Science.gov (United States)

    Choudhury, Mudra; Ramsey, Stephen A

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available.

  5. Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles

    Science.gov (United States)

    Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza

    2012-11-01

    The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors.

  6. Cell-Type-Specific Differentiation and Molecular Profiles in Skin Transplantation: Implication of Medical Approach for Genetic Skin Diseases

    Directory of Open Access Journals (Sweden)

    Noritaka Oyama

    2011-01-01

    Full Text Available Skin is highly accessible and valuable organ, which holds promise to accelerate the understanding of future medical innovation in association with skin transplantation, engineering, and wound healing. In skin transplantation biology, multistage and multifocal damages occur in both grafted donor and perilesional host skin and need to be repaired properly for the engraftment and maintenance of characteristic skin architecture. These local events are more unlikely to be regulated by the host immunity, because human skin transplantation has accomplished the donor skin engraftment onto the immunocompromised or immunosuppressive animals. Recent studies have emerged the importance of α-smooth muscle actin- (SMA- positive myofibroblasts, via stage- and cell-specific contribution of TGFβ, PDGF, ET-1, CCN-2 signalling pathways, and mastocyte-derived mediators (e.g., histamine and tryptase, for the functional reorganisation of the grafted skin. Moreover, particular cell lineages from bone marrow (BM cells have been shown to harbour the diferentiation capacity into multiple skin cell phenotypes, including epidermal keratinocytes and dermal endothelial cells and pericytes, undercontrolled by chemokines or cytokines. From a dermatological viewpoint, we review the recent update of cell-type- and molecular-specific action associated with reconstitution of the grafted skin and also focus on the novel application of BM transplantation medicine in genetic skin diseases.

  7. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.

    Science.gov (United States)

    Kuda, Ondrej; Rombaldova, Martina; Janovska, Petra; Flachs, Pavel; Kopecky, Jan

    2016-01-15

    Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA.

  8. A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis

    Science.gov (United States)

    2013-01-01

    Background About 80% of today’s land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. Results In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM

  9. Cell-type-specific circuit connectivity of hippocampal CA1 revealed through Cre-dependent rabies tracing.

    Science.gov (United States)

    Sun, Yanjun; Nguyen, Amanda Q; Nguyen, Joseph P; Le, Luc; Saur, Dieter; Choi, Jiwon; Callaway, Edward M; Xu, Xiangmin

    2014-04-10

    We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC), the medial septum (MS), and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.

  10. Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

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    Zigman Warren B

    2006-03-01

    Full Text Available Abstract Background Down syndrome (DS is caused by trisomy 21 (+21, but the aberrations in gene expression resulting from this chromosomal aneuploidy are not yet completely understood. Methods We used oligonucleotide microarrays to survey mRNA expression in early- and late-passage control and +21 fibroblasts and mid-gestation fetal hearts. We supplemented this analysis with northern blotting, western blotting, real-time RT-PCR, and immunohistochemistry. Results We found chromosome 21 genes consistently over-represented among the genes over-expressed in the +21 samples. However, these sets of over-expressed genes differed across the three cell/tissue types. The chromosome 21 gene MX1 was strongly over-expressed (mean 16-fold in senescent +21 fibroblasts, a result verified by northern and western blotting. MX1 is an interferon target gene, and its mRNA was induced by interferons present in +21 fibroblast conditioned medium, suggesting an autocrine loop for its over-expression. By immunohistochemistry the p78MX1 protein was induced in lesional tissue of alopecia areata, an autoimmune disorder associated with DS. We found strong over-expression of the purine biosynthesis gene GART (mean 3-fold in fetal hearts with +21 and verified this result by northern blotting and real-time RT-PCR. Conclusion Different subsets of chromosome 21 genes are over-expressed in different cell types with +21, and for some genes this over-expression is non-linear (>1.5X. Hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS, and abnormal purine metabolism should be investigated for a potential role in cardiac defects.

  11. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G

    2006-01-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral...... delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes...

  12. Research data supporting “Towards Cellular Sieving: Exploring the Limits of Scaffold Accessibility for Cell-Type Specific Invasion”

    OpenAIRE

    Ashworth, Jennifer C; Mehr, Marco; Buxton, Paul G.; Best, Serena M.; Cameron, Ruth E.

    2016-01-01

    Zip folder containing sample reconstructed Micro-CT scans (.tif files) from each scaffold condition. Image pixel size is 3.1 μm. Each image plane is perpendicular to the direction of cell invasion (i.e. parallel to the seeding plane). Two Microsoft Excel files, containing the raw measurements of pore size and of L and d (as defined in the manuscript) for calculation of percolation diameter. Units are in pixels unless specified. Zip folder containing raw images (.png files) of each scaffold se...

  13. Cell-Type-Specific Circuit Connectivity of Hippocampal CA1 Revealed through Cre-Dependent Rabies Tracing

    Directory of Open Access Journals (Sweden)

    Yanjun Sun

    2014-04-01

    Full Text Available We developed and applied a Cre-dependent, genetically modified rabies-based tracing system to map direct synaptic connections to specific CA1 neuron types in the mouse hippocampus. We found common inputs to excitatory and inhibitory CA1 neurons from CA3, CA2, the entorhinal cortex (EC, the medial septum (MS, and, unexpectedly, the subiculum. Excitatory CA1 neurons receive inputs from both cholinergic and GABAergic MS neurons, whereas inhibitory neurons receive a great majority of inputs from GABAergic MS neurons. Both cell types also receive weaker input from glutamatergic MS neurons. Comparisons of inputs to CA1 PV+ interneurons versus SOM+ interneurons showed similar strengths of input from the subiculum, but PV+ interneurons received much stronger input than SOM+ neurons from CA3, the EC, and the MS. Thus, rabies tracing identifies hippocampal circuit connections and maps how the different input sources to CA1 are distributed with different strengths on each of its constituent cell types.

  14. The Stage- and Cell Type-Specific Localization of Fragile X Mental Retardation Protein in Rat Ovaries.

    Science.gov (United States)

    Takahashi, Noriyuki; Tarumi, Wataru; Itoh, Masanori T; Ishizuka, Bunpei

    2015-12-01

    Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

  15. Mapping mammalian cell-type-specific transcriptional regulatory networks using KD-CAGE and ChIP-seq data in the TC-YIK cell line.

    Directory of Open Access Journals (Sweden)

    Marina eLizio

    2015-11-01

    Full Text Available Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5, we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD, we then used Cap Analysis of Gene Expression (CAGE to identify thousands of their targets genome-wide (KD-CAGE. The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN, and ISL1 and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6 and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 1kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e. TF-TF only, NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1 and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6 and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting

  16. Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line.

    Science.gov (United States)

    Lizio, Marina; Ishizu, Yuri; Itoh, Masayoshi; Lassmann, Timo; Hasegawa, Akira; Kubosaki, Atsutaka; Severin, Jessica; Kawaji, Hideya; Nakamura, Yukio; Suzuki, Harukazu; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R

    2015-01-01

    Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting Ch

  17. Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

    Science.gov (United States)

    Vasilopoulou, E.; Loubière, L.S.; Lash, G.E.; Ohizua, O.; McCabe, C.J.; Franklyn, J.A.; Kilby, M.D.; Chan, S.Y.

    2014-01-01

    , TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote

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

    Directory of Open Access Journals (Sweden)

    Ronna Hertzano

    2011-09-01

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

  19. Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

    Directory of Open Access Journals (Sweden)

    Deutsch Eric W

    2008-05-01

    Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

  20. Regional and cell-type-specific effects of DAMGO on striatal D1 and D2 dopamine receptor-expressing medium-sized spiny neurons

    Directory of Open Access Journals (Sweden)

    Christopher J Evans

    2012-03-01

    Full Text Available The striatum can be divided into the DLS (dorsolateral striatum and the VMS (ventromedial striatum, which includes NAcC (nucleus accumbens core and NAcS (nucleus accumbens shell. Here, we examined differences in electrophysiological properties of MSSNs (medium-sized spiny neurons based on their location, expression of DA (dopamine D1/D2 receptors and responses to the μ-opioid receptor agonist, DAMGO {[D-Ala2-MePhe4-Gly(ol5]enkephalin}. The main differences in morphological and biophysical membrane properties occurred among striatal sub-regions. MSSNs in the DLS were larger, had higher membrane capacitances and lower Rin (input resistances compared with cells in the VMS. RMPs (resting membrane potentials were similar among regions except for D2 cells in the NAcC, which displayed a significantly more depolarized RMP. In contrast, differences in frequency of spontaneous excitatory synaptic inputs were more prominent between cell types, with D2 cells receiving significantly more excitatory inputs than D1 cells, particularly in the VMS. Inhibitory inputs were not different between D1 and D2 cells. However, MSSNs in the VMS received more inhibitory inputs than those in the DLS. Acute application of DAMGO reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents, but the effect was greater in the VMS, in particular in the NAcS, where excitatory currents from D2 cells and inhibitory currents from D1 cells were inhibited by the largest amount. DAMGO also increased cellular excitability in the VMS, as shown by reduced threshold for evoking APs (action potentials. Together the present findings help elucidate the regional and cell-type-specific substrate of opioid actions in the striatum and point to the VMS as a critical mediator of DAMGO effects.

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

    Directory of Open Access Journals (Sweden)

    Hironori Waki

    2011-10-01

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

  2. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    Science.gov (United States)

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  3. Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

    Science.gov (United States)

    Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

    2016-09-01

    Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

  4. p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

    Science.gov (United States)

    Hattori, Hiroyoshi; Janky, Rekin's; Nietfeld, Wilfried; Aerts, Stein; Madan Babu, M; Venkitaraman, Ashok R

    2014-01-01

    The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

  5. Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis.

    Science.gov (United States)

    Rubio-Peña, Karinna; Fontrodona, Laura; Aristizábal-Corrales, David; Torres, Silvia; Cornes, Eric; García-Rodríguez, Francisco J; Serrat, Xènia; González-Knowles, David; Foissac, Sylvain; Porta-De-La-Riva, Montserrat; Cerón, Julián

    2015-12-01

    Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. Interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability.

  6. Molecular analysis of cell type-specific gene expression profile during mouse spermatogenesis by laser microdissection and qRT-PCR.

    Science.gov (United States)

    Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Moley, Kelle H

    2013-03-01

    Laser microdissection (LMD) is a selective cell isolation technique that enables the separation of desired homogenous cell subpopulations from complex tissues such as the testes under direct microscopic visualization. The LMD accompanied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) represents an indispensable tool in quantifying messenger RNA (mRNA) expression among defined cell populations. Gene expression is temporally and spatially regulated at 3 sequential phases of mitotic, meiotic, and postmeiotic stages of spermatogenesis. The present study demonstrates a short modified LMD protocol based upon hematoxylin and eosin (H&E) staining. Stage-specific LMD success was validated by the use of mRNA profiling of "marker genes" which are conserved across species and are known to be differentially expressed during spermatogenesis. Magea4, Hspa2, Cox6b2, Tnp1, Prm1, and Prm2 are used to differentiate among the microdissected cell populations, namely spermatogonia (group I), spermatocytes (group II), round and condensing spermatids (group III), and elongated and condensed spermatids (group IV), respectively. The LMD combined with qRT-PCR is further extended to assess the cell stage-specific distribution of selected stress response genes such as Hsp90aa1, Gpx4, Ucp2, Sod1, and Sod2. The germ cell-specific mRNA profiles are suitably complemented by Western blot of the LMD samples, immunohistochemistry, and confocal localization of the corresponding proteins. The current study suggests that LMD can successfully isolate cell subpopulations from the complex tissues of the testes; and establish cell stage-specific basal expression patterns of selected stress response genes and proteins. It is our hypothesis that the baseline expression of stress response genes will differ by cell stage to create discrete stage-specific vulnerabilities to reproductive toxicants.

  7. Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns

    DEFF Research Database (Denmark)

    Leroux, Olivier; Sørensen, Iben; Marcus, Susan E.;

    2015-01-01

    plants, ferns have been largely neglected in cell wall comparative studies. Results: To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species...... across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection...... in ground tissues. Mannan epitopes were found to be associated with the development of mechanical tissues. We provided the first evidence for the presence of MLG in leptosporangiate ferns. Conclusions: The data sets indicate that cell wall diversity in land plants is multifaceted and that matrix glycan...

  8. Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns

    DEFF Research Database (Denmark)

    Leroux, Olivier; Sørensen, Iben; Marcus, Susan E.;

    2015-01-01

    across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection...

  9. Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Philipp Gierok

    Full Text Available Staphylococcus aureus alpha-toxin (Hla is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o- under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml of recombinant Hla (rHla in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o- cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

  10. The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response

    Directory of Open Access Journals (Sweden)

    Stenman Göran

    2008-07-01

    Full Text Available Abstract Background FUS, EWS and TAF15 are structurally similar multifunctional proteins that were first discovered upon characterization of fusion oncogenes in human sarcomas and leukemias. The proteins belong to the FET (previously TET family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. In the present study, we investigated the expression and cellular localization of FET proteins in multiple human tissues and cell types. Results FUS, EWS and TAF15 were expressed in both distinct and overlapping patterns in human tissues. The three proteins showed almost ubiquitous nuclear expression and FUS and TAF15 were in addition present in the cytoplasm of most cell types. Cytoplasmic EWS was more rarely detected and seen mainly in secretory cell types. Furthermore, FET expression was downregulated in differentiating human embryonic stem cells, during induced differentiation of neuroblastoma cells and absent in terminally differentiated melanocytes and cardiac muscle cells. The FET proteins were targeted to stress granules induced by heat shock and oxidative stress and FUS required its RNA-binding domain for this translocation. Furthermore, FUS and TAF15 were detected in spreading initiation centers of adhering cells. Conclusion Our results point to cell-specific expression patterns and functions of the FET proteins rather than the housekeeping roles inferred from earlier studies. The localization of FET proteins to stress granules suggests activities in translational regulation during stress conditions. Roles in central processes such as stress response, translational control and adhesion may explain the FET proteins frequent involvement in human cancer.

  11. Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

    Science.gov (United States)

    Morelli, A E; Larregina, A T; Smith-Arica, J; Dewey, R A; Southgate, T D; Ambar, B; Fontana, A; Castro, M G; Lowenstein, P R

    1999-03-01

    Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

  12. High Glutathione and Glutathione Peroxidase-2 Levels Mediate Cell-Type-Specific DNA Damage Protection in Human Induced Pluripotent Stem Cells

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    Benjamin Dannenmann

    2015-05-01

    Full Text Available Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs, we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.

  13. Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein

    NARCIS (Netherlands)

    Lengerer, Birgit; Pjeta, Robert; Wunderer, Julia; Rodrigues, Marcelo; Arbore, Roberto; Schaerer, Lukas; Berezikov, Eugene; Hess, Michael W.; Pfaller, Kristian; Egger, Bernhard; Obwegeser, Sabrina; Salvenmoser, Willi; Ladurner, Peter

    2014-01-01

    Background: Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an an

  14. Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

    DEFF Research Database (Denmark)

    Gotfryd, Kamil; Skladchikova, Galina; Lepekhin, Eugene E

    2010-01-01

    ABSTRACT: BACKGROUND: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods: The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell....../2 phosphorylation are also important for the anti-cancer properties of VPA....

  15. Cell-type-specific Jumonji histone demethylase gene expression in the healthy rat CNS: detection by a novel flow cytometry method

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    Stephanie M.C. Smith

    2014-05-01

    Full Text Available Our understanding of how histone demethylation contributes to the regulation of basal gene expression in the brain is largely unknown in any injury model, and especially in the healthy adult brain. Although Jumonji genes are often regulated transcriptionally, cell-specific gene expression of Jumonji histone demethylases in the brain remains poorly understood. Thus, in the present study we profiled the mRNA levels of 26 Jumonji genes in microglia (CD11b+, neurons (NeuN+ and astrocytes (GFAP+ from the healthy adult rat brain. We optimized a method combining a mZBF (modified zinc-based fixative and FCM (flow cytometry to simultaneously sort cells from non-transgenic animals. We evaluated cell-surface, intracellular and nuclear proteins, including histones, as well as messenger- and micro-RNAs in different cell types simultaneously from a single-sorted sample. We found that 12 Jumonji genes were differentially expressed between adult microglia, neurons and astrocytes. While JMJD2D was neuron-restricted, PHF8 and JMJD1C were expressed in all three cell types although the expression was highest in neurons. JMJD3 and JMJD5 were expressed in all cell types, but were highly enriched in microglia; astrocytes had the lowest expression of UTX and JHDM1D. Levels of global H3K27 (H3 lysine 27 methylation varied among cell types and appeared to be lowest in microglia, indicating that differences in basal gene expression of specific Jumonji histone demethylases may contribute to cell-specific gene expression in the CNS (central nervous system. This multiparametric technique will be valuable for simultaneously assaying chromatin modifications and gene regulation in the adult CNS.

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

    Directory of Open Access Journals (Sweden)

    Manoj B Menon

    2014-08-01

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

  17. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    Directory of Open Access Journals (Sweden)

    Sheng Hu

    Full Text Available DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  18. Dynamics and Cell-Type Specificity of the DNA Double-Strand Break Repair Protein RecN in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120.

    Science.gov (United States)

    Hu, Sheng; Wang, Jinglan; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2015-01-01

    DNA replication and repair are two fundamental processes required in life proliferation and cellular defense and some common proteins are involved in both processes. The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is capable of forming heterocysts for N2 fixation in the absence of a combined-nitrogen source. This developmental process is intimately linked to cell cycle control. In this study, we investigated the localization of the DNA double-strand break repair protein RecN during key cellular events, such as chromosome damaging, cell division, and heterocyst differentiation. Treatment by a drug causing DNA double-strand breaks (DSBs) induced reorganization of the RecN focus preferentially towards the mid-cell position. RecN-GFP was absent in most mature heterocysts. Furthermore, our results showed that HetR, a central player in heterocyst development, was involved in the proper positioning and distribution of RecN-GFP. These results showed the dynamics of RecN in DSB repair and suggested a differential regulation of DNA DSB repair in vegetative cell and heterocysts. The absence of RecN in mature heterocysts is compatible with the terminal nature of these cells.

  19. Cell Type-Specific Delivery of RNAi by Ligand-Functionalized Curdlan Nanoparticles: Balancing the Receptor Mediation and the Charge Motivation.

    Science.gov (United States)

    Wu, Yinga; Cai, Jia; Han, Jingfen; Baigude, Huricha

    2015-09-30

    Tissue-specific delivery of therapeutic RNAi has great potential for clinical applications. Receptor-mediated endocytosis plays a crucial role in targeted delivery of biotherapeutics including short interfering RNA (siRNA). Previously we reported a novel Curdlan-based nanoparticle for intracellular delivery of siRNA. Here we designed a nanoparticle based on ligand-functionalized Curdlan. Disaccharides were site-specifically conjugated to 6-deoxy-6-amino Curdlan, and the cell line specificity, cellular uptake, cytotoxicity, and siRNA delivery efficiency of the corresponding disaccharide-modified 6-deoxy-6-amino-Curdlan were investigated. Observation by fluorescence microscopy as well as flow cytometry showed that galactose-containing Curdlan derivatives delivered fluorescently labeled short nucleic acid to HepG2 cells expressing ASGPR receptor but not in other cells lacking surface ASGPR protein. Moreover, highly galactose-substituted Curdlan derivatives delivered siRNA specifically to ASGPR-expressing cells and induced RNAi activities, silencing endogenous GAPDH gene expression. Our data demonstrated that galactose-functionalized 6-deoxy-6-amino-Curdlan is a promising carrier for short therapeutic nucleic acids for clinical applications.

  20. Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP.

    Science.gov (United States)

    Ruoff, Rachel; Katsara, Olga; Kolupaeva, Victoria

    2016-07-05

    Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

  1. A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

    Science.gov (United States)

    Serrano, Mónica; Kint, Nicolas; Pereira, Fátima C.; Saujet, Laure; Boudry, Pierre; Dupuy, Bruno; Martin-Verstraete, Isabelle

    2016-01-01

    The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development. PMID:27631621

  2. Mutations in the Caenorhabditis elegans let-23 EGFR-like gene define elements important for cell-type specificity and function.

    Science.gov (United States)

    Aroian, R V; Lesa, G M; Sternberg, P W

    1994-01-01

    The Caenorhabditis elegans let-23 gene is a genetically characterized member of the epidermal growth factor receptor (EGFR) tyrosine kinase family. Mutations in let-23 can produce five phenotypes in the nematode. Alleles of let-23 include null alleles, reduction-of-function alleles and alleles that disrupt function in some cell types and not others. We have sequenced some of these mutations to identify sequences and regions important for overall let-23 function and for let-23 function in specific cell types. Our data indicate that in vivo, the receptor's C-terminus can be partitioned into at least three domains that each contribute to receptor function in different cell types. In particular, we find distinct domains that mediate hermaphrodite fertility and vulval induction. Our data also demonstrate for the first time that a single, conserved residue in the ligand binding domain is critical for function in vivo and that mutations in the extracellular cysteines characteristic of the EGFR family can lead to a partial or a complete reduction of receptor function. Images PMID:8313880

  3. Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

    Science.gov (United States)

    Ron, Mily; Kajala, Kaisa; Pauluzzi, Germain; Wang, Dongxue; Reynoso, Mauricio A; Zumstein, Kristina; Garcha, Jasmine; Winte, Sonja; Masson, Helen; Inagaki, Soichi; Federici, Fernán; Sinha, Neelima; Deal, Roger B; Bailey-Serres, Julia; Brady, Siobhan M

    2014-10-01

    Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

  4. Cell-type-specific expression and regulation of a c-fos-NGF fusion gene in neurons and astrocytes of transgenic mice.

    Science.gov (United States)

    Onténiente, B; Horellou, P; Neveu, I; Makeh, I; Suzuki, F; Bourdet, C; Grimber, G; Colin, P; Brachet, P; Mallet, J

    1994-02-01

    A mouse line transgenic for nerve growth factor (NGF) was developed using the mouse prepro-NGF cDNA inserted within a plasmid containing the proximal region (-10 to -550 bp) of the c-fos promoter and the transcription termination and polyadenylation signals of the rabbit beta-globin gene. No significant modification of gross behavior or central nervous system anatomy was detected in adult animals as assessed by immunohistochemistry and in situ hybridization for NGF and choline acetyltransferase. The expression of the transgene and the possible regulation of its expression by agents acting on the promoter were investigated in vitro. Despite the presence of an additional pool of NGF mRNA specific to the transgene, basal levels of NGF in the supernatant of transgenic astrocytes were similar to normal ones. On the other hand, transgenic neurons spontaneously synthesized and released levels of NGF two to three times higher than normal neurons, while mRNA levels were barely detectable by conventional Northern blotting. The tissue-specificity of NGF expression was respected, with higher levels in hippocampal than neocortical neurons. Increases of NGF mRNA by agents acting on the promoter could be observed in normal and transgenic astrocytes only after inhibition of the protein synthesis by cycloheximide, suggesting a similar rapid turnover of normal and transgenic transcripts. Cyclic AMP agonists specifically increased the secretion of NGF protein by transgenic astrocytes and neurons, while activators of the protein kinase C had a similar effect on transgenic and normal cells. Differences between amounts of NGF secreted by neurons and astrocytes with regards to their respective content in mRNA suggest that transgenic transcripts are subject to normal cell- and tissue-specific post-transcriptional regulations. Agents acting on the c-fos promoter through the protein kinase C or cyclic AMP routes differentially increased the secretion of NGF by transgenic astrocytes or

  5. The M-current contributes to high threshold membrane potential oscillations in a cell type-specific way in the pedunculopontine nucleus of mice

    Directory of Open Access Journals (Sweden)

    Csilla eBordas

    2015-04-01

    Full Text Available The pedunculopontine nucleus is known as a cholinergic nucleus of the reticular activating system, participating in regulation of sleep and wakefulness. Besides cholinergic neurons, it consists of GABAergic and glutamatergic neurons as well. According to classical and recent studies, more subgroups of neurons were defined. Groups based on the neurotransmitter released by a neuron are not homogenous, but can be further subdivided.The PPN neurons do not only provide cholinergic and non-cholinergic inputs to several subcortical brain areas but they are also targets of cholinergic and other different neuromodulatory actions. Although cholinergic neuromodulation has been already investigated in the nucleus, one of its characteristic targets, the M-type potassium current has not been described yet.Using slice electrophysiology, we provide evidence in the present work that cholinergic neurons possess M-current, whereas GABAergic neurons lack it. The M-current contributes to certain functional differences of cholinergic and GABAergic neurons, as spike frequency adaptation, action potential firing frequency or the amplitude difference of medium afterhyperpolarizations. Furthermore, we showed that high threshold membrane potential oscillation with high power, around 20 Hz frequency is a functional property of almost all cholinergic cells, whereas GABAergic neurons have only low amplitude oscillations. Blockade of the M-current abolished the oscillatory activity at 20 Hz, and largely diminished it at other frequencies.Taken together, the M-current seems to be characteristic for PPN cholinergic neurons. It provides a possibility for modulating gamma band activity of these cells, thus contributing to neuromodulatory regulation of the reticular activating system.

  6. Coupling the GAL4 UAS system with alcR for versatile cell type-specific chemically inducible gene expression in Arabidopsis.

    Science.gov (United States)

    Sakvarelidze, Lali; Tao, Zheng; Bush, Max; Roberts, Gethin R; Leader, David J; Doonan, John H; Rawsthorne, Stephen

    2007-07-01

    The Aspergillus alc regulon encodes a transcription factor, ALCR, which regulates transcription from cognate promoters such as alcA(p). In the presence of suitable chemical inducers, ALCR activates gene expression from alcA(p). The alc regulon can be transferred to other species and can be used to control the expression of reporter, metabolic and developmental genes in response to low-level ethanol exposure. In this paper, we describe a versatile system for targeting the alc regulon to specific cell types in Arabidopsis by driving ALCR expression from the GAL4 upstream activator sequence (UAS). Large numbers of Arabidopsis lines are available in which GAL4 is expressed in a variety of spatial patterns and, in turn, drives the expression of any gene cloned downstream of the UAS. We have used a previously characterized line that directs gene expression to the endosperm to demonstrate spatially restricted ethanol-inducible gene expression. We also show that the domain of inducible gene expression can easily be altered by crossing the UAS::ALCR cassette into different driver lines. We conclude that this gene switch can be used to drive gene expression in a highly responsive, but spatially restricted, manner.

  7. Expression of the Ly-6 family proteins Lynx1 and Ly6H in the rat brain is compartmentalized, cell-type specific, and developmentally regulated

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Cinar, Betül; Jensen, Majbrit Myrup

    2014-01-01

    regarding the distribution and developmental regulation of these proteins in the brain. We use protein cross-linking and synaptosomal fractions to demonstrate that the Ly-6 proteins Lynx1 and Ly6H are membrane-bound proteins in the brain, which are present on the cell surface and localize to synaptic...... compartments. We further estimate the amount of Lynx1 in the rat cortex using known amounts of a heterologously expressed soluble Lynx1 variant (ws-Lynx1) to be approximately 8.6 ng/μg total protein, which is in line with the concentrations of ws-Lynx1 required to affect nAChR function. In addition, we...... demonstrate that Lynx1 and Ly6H are expressed in cultured neurons, but not cultured micro- or astroglial cultures. In addition, Lynx1, but not Ly6H was detected in the CSF. Finally, we show that the Ly-6 proteins Lynx1, Lynx2, Ly6H, and PSCA, display distinct expression patterns during postnatal development...

  8. Cell-type specific deletion of GABA(A)α1 in corticotropin-releasing factor-containing neurons enhances anxiety and disrupts fear extinction.

    Science.gov (United States)

    Gafford, Georgette M; Guo, Ji-Dong; Flandreau, Elizabeth I; Hazra, Rimi; Rainnie, Donald G; Ressler, Kerry J

    2012-10-02

    Corticotropin-releasing factor (CRF) is critical for the endocrine, autonomic, and behavioral responses to stressors, and it has been shown to modulate fear and anxiety. The CRF receptor is widely expressed across a variety of cell types, impeding progress toward understanding the contribution of specific CRF-containing neurons to fear dysregulation. We used a unique CRF-Cre driver transgenic mouse line to remove floxed GABA(A)α1 subunits specifically from CRF neurons [CRF-GABA(A)α1 KO]. This process resulted in mice with decreased GABA(A)α1 expression only in CRF neurons and increased CRF mRNA within the amygdala, bed nucleus of the stria terminalis (BNST) and paraventricular nucleus of the hypothalamus. These mice show normal locomotor and pain responses and no difference in depressive-like behavior or Pavlovian fear conditioning. However, CRF-GABA(A)α1 KO increased anxiety-like behavior and impaired extinction of conditioned fear, coincident with an increase in plasma corticosterone concentration. These behavioral impairments were rescued with systemic or BNST infusion of the CRF antagonist R121919. Infusion of Zolpidem, a GABA(A)α1-preferring benzodiazepine-site agonist, into the BNST of the CRF-GABA(A)α1 KO was ineffective at decreasing anxiety. Electrophysiological findings suggest a disruption in inhibitory current may play a role in these changes. These data indicate that disturbance of CRF containing GABA(A)α1 neurons causes increased anxiety and impaired fear extinction, both of which are symptoms diagnostic for anxiety disorders, such as posttraumatic stress disorder.

  9. The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants.

    Science.gov (United States)

    Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

    2014-01-01

    In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

  10. The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

    Directory of Open Access Journals (Sweden)

    Madlen eNietzsche

    2014-02-01

    Full Text Available In plants, SNF1-related kinase (SnRK1 responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11 of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins can share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

  11. The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

    Science.gov (United States)

    Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

    2014-01-01

    In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation. PMID:24600465

  12. Visualizing T cell migration in-situ

    Directory of Open Access Journals (Sweden)

    Alexandre P Benechet

    2014-07-01

    Full Text Available Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid tissues. The structure of secondary lymphoid organs regulates immune responses by promoting optimal cell-cell and cell-extracellular matrix interactions. Naïve T cells are initially activated by antigen presenting cells in secondary lymphoid organs. Following priming, effector T cells migrate to the site of infection to exert their functions. Majority of the effector cells die while a small population of antigen specific T cells persist as memory cells in distinct anatomical locations. The persistence and location of memory cells in lymphoid and non-lymphoid tissues is critical to protect the host from re-infection. The localization of memory T cells is carefully regulated by several factors including the highly organized secondary lymphoid structure, the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands. This balance between the anatomy and the ordered expression of cell surface and soluble proteins regulates the subtle choreography of T cell migration. In recent years, our understanding of cellular dynamics of T cells has been advanced by the development of new imaging techniques allowing in-situ visualization of T cell responses. Here we review the past and more recent studies that have utilized sophisticated imaging technologies to investigate the migration dynamics of naive, effector and memory T cells.

  13. Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

    Science.gov (United States)

    Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

    2000-01-01

    We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

  14. Prevalence of type-specific HPV infection in Uruguay.

    Science.gov (United States)

    Berois, Nora; Heard, Isabelle; Fort, Zoraida; Alonso, Rafael; Sica, Adela; Moerzinger, Patricia; Rodriguez, Guillermo; Sancho-Garnier, Hélène; Osinaga, Eduardo; Favre, Michel

    2014-04-01

    The aim of this work was to describe the prevalence of type-specific Human papillomavirus (HPV) infection in women attending organized cervical cancer screening program in Uruguay. Nine hundred sixty-five liquid cervical cell samples obtained after collection of cervical smears for cytology were assessed for HPV DNA using the Papillocheck system (Greiner BioOne). The overall prevalence of High-Risk (HR) HPV infections was 20.8% and increased from 16.5% in women with normal cytology to 93.3% in HSIL. Prevalence of HPV 16 and/or 18 was 6.3% and HPV 16 was the most prevalent genotype in normal cytology (3.6%). The five most prevalent genotypes were HPV 16, 31, 51, 56, and 39. The overall prevalence peaked below age 30. This study provides essential baseline information at national level on type-specific HPV prevalence in Uruguay before the introduction of HPV vaccination. It documents the current prevalence of each of the oncogenic genotypes in a population attending cervical cancer screening program, suggesting that at least 64.7% of high risk lesions are potentially preventable by available HPV vaccines, and possibly augmentable if cross-protection against non-vaccine HPV types 31, 33, and 45 is confirmed.

  15. Visualizing Without Vision at the Microscale: Students With Visual Impairments Explore Cells With Touch

    Science.gov (United States)

    Jones, M. Gail; Minogue, James; Oppewal, Tom; Cook, Michelle P.; Broadwell, Bethany

    2006-12-01

    Science instruction is typically highly dependent on visual representations of scientific concepts that are communicated through textbooks, teacher presentations, and computer-based multimedia materials. Little is known about how students with visual impairments access and interpret these types of visually-dependent instructional materials. This study explored the efficacy of new haptic (simulated tactile feedback and kinesthetics) instructional technology for teaching cell morphology and function to middle and high school students with visual impairments. The study examined students' prior experiences learning about the cell and cell functions in classroom instruction, as well as how haptic feedback technology impacted students' awareness of the 3-D nature of an animal cell, the morphology and function of cell organelles, and students' interest in the haptic technology as an instructional tool. Twenty-one students with visual impairment participated in the study. Students explored a tactile model of the cell with a haptic point probe that allowed them to feel the cell and its organelles. Results showed that students made significant gains in their ability to identify cell organelles and found the technology to be highly interesting as an instructional tool. The need for additional adaptive technology for students with visual impairments is discussed.

  16. Neuronal survival in the brain: neuron type-specific mechanisms.

    Science.gov (United States)

    Pfisterer, Ulrich; Khodosevich, Konstantin

    2017-03-02

    Neurogenic regions of mammalian brain produce many more neurons that will eventually survive and reach a mature stage. Developmental cell death affects both embryonically produced immature neurons and those immature neurons that are generated in regions of adult neurogenesis. Removal of substantial numbers of neurons that are not yet completely integrated into the local circuits helps to ensure that maturation and homeostatic function of neuronal networks in the brain proceed correctly. External signals from brain microenvironment together with intrinsic signaling pathways determine whether a particular neuron will die. To accommodate this signaling, immature neurons in the brain express a number of transmembrane factors as well as intracellular signaling molecules that will regulate the cell survival/death decision, and many of these factors cease being expressed upon neuronal maturation. Furthermore, pro-survival factors and intracellular responses depend on the type of neuron and region of the brain. Thus, in addition to some common neuronal pro-survival signaling, different types of neurons possess a variety of 'neuron type-specific' pro-survival constituents that might help them to adapt for survival in a certain brain region. This review focuses on how immature neurons survive during normal and impaired brain development, both in the embryonic/neonatal brain and in brain regions associated with adult neurogenesis, and emphasizes neuron type-specific mechanisms that help to survive for various types of immature neurons. Importantly, we mainly focus on in vivo data to describe neuronal survival specifically in the brain, without extrapolating data obtained in the PNS or spinal cord, and thus emphasize the influence of the complex brain environment on neuronal survival during development.

  17. Optogenetics Advances in Primate Visual Pathway.

    Science.gov (United States)

    Jazayeri, Mehrdad; Remington, Evan

    2016-04-06

    In this issue of Neuron, Klein et al. (2016) used cell-type-specific optogenetics and electrical microstimulation to characterize the koniocellular geniculocortical projections in nonhuman primates. Their work offers a powerful platform for refining our understanding of the mechanisms of visual information processing in the lateral geniculate nucleus and primary visual cortex.

  18. Atypical visual loss in giant cell arteritis

    DEFF Research Database (Denmark)

    Thystrup, Jan Deichmann; Knudsen, G M; Mogensen, A M

    1994-01-01

    in the terminal stage of his disease due to bilateral occipital cortex infarctions, verified by CT-scan. Autopsy revealed involvement of several intracranial arteries. In case No. 2 there was severe unilateral visual loss and cotton-wool exudates in both eyes. Central vision recovered after corticosteroid therapy......; in our experience this is unusual. In case No. 3 irreversible unilateral visual loss was typical for GCA, but the association with polyneuropathy unique. Neurological remission coincided with systemic corticosteroid therapy....

  19. [Visual hallucinations and giant cell arteritis: the Charles Bonnet syndrome].

    Science.gov (United States)

    Bloch, J; Morell-Dubois, S; Koch, E; Launay, D; Maillard-Lefebvre, H; Buchdahl, A-L; Hachulla, E; Rouland, J-F; Hatron, P-Y; Lambert, M

    2011-12-01

    In patients with visual hallucinations, diagnostic strategy is unclearly codified. In patients known to have giant cell arteritis, the main diagnostic assumption is disease relapse. Indeed, this should lead to rapid corticosteroid therapy. However, the Charles Bonnet syndrome, that is a poorly known etiology of visual hallucinations usually observed in elderly people, should be part of the differential diagnosis. We report a 87-year-old woman, with a 2-year history of giant cell arteritis who was admitted with an acute onset of visual hallucinations and who met all the criteria for Charles Bonnet syndrome.

  20. The cell-type specific uptake of polymer-coated or micelle-embedded QDs and SPIOs does not provoke an acute pro-inflammatory response in the liver

    Directory of Open Access Journals (Sweden)

    Markus Heine

    2014-09-01

    Full Text Available Semiconductor quantum dots (QD and superparamagnetic iron oxide nanocrystals (SPIO have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene (PMAOD. The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr-/- as well as Apoe-/- mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα or chemokine (C-X-C motif ligand 10 (Cxcl10 indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications.

  1. Visualization

    OpenAIRE

    Balon, Andreja

    1990-01-01

    The present thesis entails the field of visualization which is divided into visualization along traditional lines and visualization in computer science. As the psychological aspect of image is of vital importance for visualization, it is shortly described in the beginning. Visualization in computer science is divided into three main fields: scientific visualization, program visualization and visual programming. An explanation and examples of approach to applications are given for each field....

  2. A single N-linked glycosylation site in the Japanese encephalitis virus prM protein is critical for cell type-specific prM protein biogenesis, virus particle release, and pathogenicity in mice.

    Science.gov (United States)

    Kim, Jeong-Min; Yun, Sang-Im; Song, Byung-Hak; Hahn, Youn-Soo; Lee, Chan-Hee; Oh, Hyun-Woo; Lee, Young-Min

    2008-08-01

    The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N(15)-X(16)-T(17), which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substituting for N(15) and/or T(17) and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an approximately 20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

  3. Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

    DEFF Research Database (Denmark)

    Rasmussen, Simon Brandtoft; Sørensen, Louise Nørgaard; Malmgaard, Lene

    2007-01-01

    and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms......Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes....... In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages...

  4. Current techniques for visualizing RNA in cells

    Science.gov (United States)

    Mannack, Lilith V.J.C.; Eising, Sebastian; Rentmeister, Andrea

    2016-01-01

    Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations. PMID:27158473

  5. Cell-fusion method to visualize interphase nuclear pore formation.

    Science.gov (United States)

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

    In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods.

  6. Visual outcome, ocular findings, and visual processing skills after allogeneic stem cell transplantation in children

    OpenAIRE

    Törnquist, Alba Lucia

    2010-01-01

    Background: Stem cell transplantation (SCT) offers a chance of cure in children with leukaemia and other life-threatening haematological, immunological, and metabolic diseases that do not respond to conventional treatment. Pre and post SCT, these children receive irradiation, and/or chemotherapy and immunosuppressive agents which like the primary disease may adversely affect the eye, the central nervous system as well as the posterior visual pathways and potentially threaten...

  7. In vivo cell biology of cancer cells visualized with fluorescent proteins.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    This chapter describes a new cell biology where the behavior of individual cells can be visualized in the living animal. Previously it has been demonstrated that fluorescent proteins can be used for whole-body imaging of metastatic tumor growth, bacterial infection, and gene expression. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of tumor-stroma interactions and especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts, and macrophages. Another example is the color coding of cells with RFP or GFP such that both cell types can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo. Mice in which the regulatory elements of the stem cell marker nestin drive GFP expression enable nascent vasculature to be visualized interacting with transplanted RFP-expressing cancer cells. Nestin-driven GFP expression can also be used to visualize hair follicle stem cells. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Highly elongated cancer cells in capillaries in living mice were observed within skin flaps. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells in the capillaries elongated to fit the width of these vessels. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

  8. IN-CELL visual examinations of K east fuel elements

    Energy Technology Data Exchange (ETDEWEB)

    Pitner, A.L.; Pyecha, T.D., Fluor Daniel Hanford

    1997-03-06

    Nine outer fuel elements were recovered from the K East Basin and transferred to a hot cell for examination. Extensive testing planned for these elements will support the process design for the Integrated Process Strategy (IPS), with emphasis on drying and conditioning behavior. Visual examinations of the fuel elements confirmed that they are appropriate to meet testing objectives to provide design guidance for IPS processing parameters.

  9. Effect of Contrast on Visual Spatial Summation in Different Cell Categories in Cat Primary Visual Cortex.

    Directory of Open Access Journals (Sweden)

    Ke Chen

    Full Text Available Multiple cell classes have been found in the primary visual cortex, but the relationship between cell types and spatial summation has seldom been studied. Parvalbumin-expressing inhibitory interneurons can be distinguished from pyramidal neurons based on their briefer action potential durations. In this study, we classified V1 cells into fast-spiking units (FSUs and regular-spiking units (RSUs and then examined spatial summation at high and low contrast. Our results revealed that the excitatory classical receptive field and the suppressive non-classical receptive field expanded at low contrast for both FSUs and RSUs, but the expansion was more marked for the RSUs than for the FSUs. For most V1 neurons, surround suppression varied as the contrast changed from high to low. However, FSUs exhibited no significant difference in the strength of suppression between high and low contrast, although the overall suppression decreased significantly at low contrast for the RSUs. Our results suggest that the modulation of spatial summation by stimulus contrast differs across populations of neurons in the cat primary visual cortex.

  10. Purification, Visualization, and Molecular Signature of Neural Stem Cells

    Science.gov (United States)

    Yu, Yuan Hong; Narayanan, Gunaseelan; Sankaran, Shvetha; Ramasamy, Srinivas; Chan, Shi Yu; Lin, Shuping; Chen, Jinmiao; Yang, Henry; Srivats, Hariharan

    2016-01-01

    Neural stem cells (NSCs) are isolated from primary brain tissue and propagated as a heterogeneous mix of cells, including neural progenitors. To date, NSCs have not been purified in vitro to allow study of their biology and utility in regenerative medicine. In this study, we identify C1qR1as a novel marker for NSCs and show that it can be used along with Lewis-X (LeX) to yield a highly purified population of NSCs. Using time-lapse microscopy, we are able to follow NSCs forming neurospheres, allowing their visualization. Finally, using single-cell polymerase chain reaction (PCR), we determine the molecular signature of NSCs. The single-cell PCR data suggest that along with the Notch and Shh pathways, the Hippo pathway plays an important role in NSC activity. PMID:26464067

  11. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    Science.gov (United States)

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  12. Immunohistochemical visualization of neurons and specific glial cells for stereological application in the porcine neocortex

    DEFF Research Database (Denmark)

    Lyck, Lise; Jelsing, Jacob; Jensen, Pia Søndergaard;

    2006-01-01

    The pig is becoming an increasingly used non-primate model in basic experimental studies of human neurological diseases. In spite of the widespread use of immunohistochemistry and cell type specific markers, the application of immunohistochemistry in the pig brain has not been systematically desc...

  13. Development of an autonomous biological cell manipulator with single-cell electroporation and visual servoing capabilities.

    Science.gov (United States)

    Sakaki, Kelly; Dechev, Nikolai; Burke, Robert D; Park, Edward J

    2009-08-01

    Studies of single cells via microscopy and microinjection are a key component in research on gene functions, cancer, stem cells, and reproductive technology. As biomedical experiments become more complex, there is an urgent need to use robotic systems to improve cell manipulation and microinjection processes. Automation of these tasks using machine vision and visual servoing creates significant benefits for biomedical laboratories, including repeatability of experiments, higher throughput, and improved cell viability. This paper presents the development of a new 5-DOF robotic manipulator, designed for manipulating and microinjecting single cells. This biological cell manipulator (BCM) is capable of autonomous scanning of a cell culture followed by autonomous injection of cells using single-cell electroporation (SCE). SCE does not require piercing the cell membrane, thereby keeping the cell membrane fully intact. The BCM features high-precision 3-DOF translational and 2-DOF rotational motion, and a second z-axis allowing top-down placement of a micropipette tip onto the cell membrane for SCE. As a technical demonstration, the autonomous visual servoing and microinjection capabilities of the single-cell manipulator are experimentally shown using sea urchin eggs.

  14. Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

    Science.gov (United States)

    Schürmann, Matthias; Frese, Natalie; Beyer, André; Heimann, Peter; Widera, Darius; Mönkemöller, Viola; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian; Gölzhäuser, Armin

    2015-11-18

    Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

  15. Visual cells remember earlier applied target: plasticity of orientation selectivity.

    Directory of Open Access Journals (Sweden)

    Narcis Ghisovan

    Full Text Available BACKGROUND: A canonical proposition states that, in mature brain, neurons responsive to sensory stimuli are tuned to specific properties installed shortly after birth. It is amply demonstrated that that neurons in adult visual cortex of cats are orientation-selective that is they respond with the highest firing rates to preferred oriented stimuli. METHODOLOGY/PRINCIPAL FINDINGS: In anesthetized cats, prepared in a conventional fashion for single cell recordings, the present investigation shows that presenting a stimulus uninterruptedly at a non-preferred orientation for twelve minutes induces changes in orientation preference. Across all conditions orientation tuning curves were investigated using a trial by trial method. Contrary to what has been previously reported with shorter adaptation duration, twelve minutes of adaptation induces mostly attractive shifts, i.e. toward the adapter. After a recovery period allowing neurons to restore their original orientation tuning curves, we carried out a second adaptation which produced three major results: (1 more frequent attractive shifts, (2 an increase of their magnitude, and (3 an additional enhancement of responses at the new or acquired preferred orientation. Additionally, we also show that the direction of shifts depends on the duration of the adaptation: shorter adaptation in most cases produces repulsive shifts, whereas adaptation exceeding nine minutes results in attractive shifts, in the same unit. Consequently, shifts in preferred orientation depend on the duration of adaptation. CONCLUSION/SIGNIFICANCE: The supplementary response improvements indicate that neurons in area 17 keep a memory trace of the previous stimulus properties, thereby upgrading cellular performance. It also highlights the dynamic nature of basic neuronal properties in adult cortex since repeated adaptations modified both the orientation tuning selectivity and the response strength to the preferred orientation. These

  16. Neuronal survival in the brain: neuron type-specific mechanisms

    DEFF Research Database (Denmark)

    Pfisterer, Ulrich Gottfried; Khodosevich, Konstantin

    2017-01-01

    Neurogenic regions of mammalian brain produce many more neurons that will eventually survive and reach a mature stage. Developmental cell death affects both embryonically produced immature neurons and those immature neurons that are generated in regions of adult neurogenesis. Removal of substantial...... a particular neuron will die. To accommodate this signaling, immature neurons in the brain express a number of transmembrane factors as well as intracellular signaling molecules that will regulate the cell survival/death decision, and many of these factors cease being expressed upon neuronal maturation...... numbers of neurons that are not yet completely integrated into the local circuits helps to ensure that maturation and homeostatic function of neuronal networks in the brain proceed correctly. External signals from brain microenvironment together with intrinsic signaling pathways determine whether...

  17. Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells

    Science.gov (United States)

    Davidson, Reshma; Liu, Yajun; Gerien, Kenneth S.; Wu, Jian-Qiu

    2017-01-01

    Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods. PMID:26519302

  18. Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells.

    Science.gov (United States)

    Davidson, Reshma; Liu, Yajun; Gerien, Kenneth S; Wu, Jian-Qiu

    2016-01-01

    Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods.

  19. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

    Science.gov (United States)

    Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

  20. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21 and Nsg-2 (P19.

    Directory of Open Access Journals (Sweden)

    Laura Digilio

    Full Text Available The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65 were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21 and Nsg-2 (P19 are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

  1. GFP-specific CD8 T cells enable targeted cell depletion and visualization of T-cell interactions.

    Science.gov (United States)

    Agudo, Judith; Ruzo, Albert; Park, Eun Sook; Sweeney, Robert; Kana, Veronika; Wu, Meng; Zhao, Yong; Egli, Dieter; Merad, Miriam; Brown, Brian D

    2015-12-01

    There are numerous cell types with scarcely understood functions, whose interactions with the immune system are not well characterized. To facilitate their study, we generated a mouse bearing enhanced green fluorescent protein (EGFP)-specific CD8(+) T cells. Transfer of the T cells into EGFP reporter animals can be used to kill EGFP-expressing cells, allowing selective depletion of desired cell types, or to interrogate T-cell interactions with specific populations. Using this system, we eliminate a rare EGFP-expressing cell type in the heart and demonstrate its role in cardiac function. We also show that naive T cells are recruited into the mouse brain by antigen-expressing microglia, providing evidence of an immune surveillance pathway in the central nervous system. The just EGFP death-inducing (Jedi) T cells enable visualization of a T-cell antigen. They also make it possible to utilize hundreds of existing EGFP-expressing mice, tumors, pathogens and other tools, to study T-cell interactions with many different cell types, to model disease states and to determine the functions of poorly characterized cell populations.

  2. Ontogenetic cell death and phagocytosis in the visual system of vertebrates.

    Science.gov (United States)

    Francisco-Morcillo, Javier; Bejarano-Escobar, Ruth; Rodríguez-León, Joaquín; Navascués, Julio; Martín-Partido, Gervasio

    2014-10-01

    Programmed cell death (PCD), together with cell proliferation, cell migration, and cell differentiation, is an essential process during development of the vertebrate nervous system. The visual system has been an excellent model on which to investigate the mechanisms involved in ontogenetic cell death. Several phases of PCD have been reported to occur during visual system ontogeny. During these phases, comparative analyses demonstrate that dying cells show similar but not identical spatiotemporally restricted patterns in different vertebrates. Additionally, the chronotopographical coincidence of PCD with the entry of specialized phagocytes in some regions of the developing vertebrate visual system suggests that factors released from degenerating cells are involved in the cell migration of macrophages and microglial cells. Contradicting this hypothesis however, in many cases the cell corpses generated during degeneration are rapidly phagocytosed by neighboring cells, such as neuroepithelial cells or Müller cells. In this review, we describe the occurrence and the sites of PCD during the morphogenesis and differentiation of the retina and optic pathways of different vertebrates, and discuss the possible relationship between PCD and phagocytes during ontogeny.

  3. Visualization and orchestration of the dynamic molecular society in cells

    Institute of Scientific and Technical Information of China (English)

    Xuebiao Yao; Guowei Fang

    2009-01-01

    @@ Visualization of specific molecules and their interactions in real space and time is essential to delineate how cellular plasticity and dynamics are achieved and orchestrated as perturbation of cellular plasticity and dynamics is detrimental to health. Elucidation of cellular dynamics requires molecular imaging at nanometer scale at millisecond resolution. The 1st International Conference on Cellular Dynamics and Chemical Biology held in Hefei, China (from 12 September to 15 September,2008) launched the quest by bringing synergism among photonics, chemistry and biology.

  4. Pineal photoreceptor cells are required for maintaining the circadian rhythms of behavioral visual sensitivity in zebrafish.

    Directory of Open Access Journals (Sweden)

    Xinle Li

    Full Text Available In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity.

  5. Cell types, circuits, and receptive fields in the mouse visual cortex.

    Science.gov (United States)

    Niell, Cristopher M

    2015-07-08

    Over the past decade, the mouse has emerged as an important model system for studying cortical function, owing to the advent of powerful tools that can record and manipulate neural activity in intact neural circuits. This advance has been particularly prominent in the visual cortex, where studies in the mouse have begun to bridge the gap between cortical structure and function, allowing investigators to determine the circuits that underlie specific visual computations. This review describes the advances in our understanding of the mouse visual cortex, including neural coding, the role of different cell types, and links between vision and behavior, and discusses how recent findings and new approaches can guide future studies.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  7. Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

    DEFF Research Database (Denmark)

    Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven;

    2010-01-01

    embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene...

  8. Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

    Directory of Open Access Journals (Sweden)

    Tadanobu Takahashi

    Full Text Available Mumps viruses show diverse cytopathic effects (CPEs of infected cells and viral plaque formation (no CPE or no plaque formation in some cases depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac, was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study, even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

  9. Retinal ganglion cell density of the black rhinoceros (Diceros bicornis): calculating visual resolution.

    Science.gov (United States)

    Pettigrew, John D; Manger, Paul R

    2008-01-01

    A single right retina from a black rhinoceros was whole mounted, stained and analyzed to determine the visual resolution of the rhinoceros, an animal with reputedly poor eyesight. A range of small (15-microm diameter) to large (100-microm diameter) ganglion cell types was seen across the retina. We observed two regions of high density of retinal ganglion cells at either end of a long, but thin, horizontal streak. The temporal specialization, which receives light from the anterior visual field, exhibited a ganglion cell density of approximately 2000/mm2, while the nasal specialization exhibited a density of approximately 1500/mm2. The retina exhibited a ganglion cell density bias toward the upper half, especially so, the upper temporal quadrant, indicating that the rhinoceros would be processing visual information from the visual field below the anterior horizon for the most part. Our calculations indicate that the rhinoceros has a visual resolution of 6 cycles/degree. While this resolution is one-tenth that of humans (60 cycles/deg) and less than that of the domestic cat (9 cycles/deg), it is comparable to that of the rabbit (6 cycles/deg), and exceeds that seen in a variety of other mammals including seals, dolphins, microbats, and rats. Thus, the reputation of the rhinoceros as a myopic, weakly visual animal is not supported by our observations of the retina. We calculate that the black rhinoceros could readily distinguish a 30 cm wide human at a distance of around 200 m given the appropriate visual background.

  10. Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle

    OpenAIRE

    Hesse, Michael; Raulf, Alexandra; Pilz, Gregor-Alexander; Haberlandt, Christian; Klein, Alexandra M; Jabs, Ronald; Zaehres, Holm; Fügemann, Christopher J.; Zimmermann, Katrin; Trebicka, Jonel; Welz, Armin; Pfeifer, Alexander; Röll, Wilhelm; Kotlikoff, Michael I.; Steinhäuser, Christian

    2012-01-01

    Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hall...

  11. 1Click1View: Interactive Visualization Methodology for RNAi Cell-Based Microscopic Screening

    Directory of Open Access Journals (Sweden)

    Lukasz Zwolinski

    2013-01-01

    Full Text Available Technological advancements are constantly increasing the size and complexity of data resulting from large-scale RNA interference screens. This fact has led biologists to ask complex questions, which the existing, fully automated analyses are often not adequate to answer. We present a concept of 1Click1View (1C1V as a methodology for interactive analytic software tools. 1C1V can be applied for two-dimensional visualization of image-based screening data sets from High Content Screening (HCS. Through an easy-to-use interface, one-click, one-view concept, and workflow based architecture, visualization method facilitates the linking of image data with numeric data. Such method utilizes state-of-the-art interactive visualization tools optimized for fast visualization of large scale image data sets. We demonstrate our method on an HCS dataset consisting of multiple cell features from two screening assays.

  12. Analysis and visualization of cell movement in the developing zebrafish brain.

    Science.gov (United States)

    Langenberg, Tobias; Dracz, Tadeusz; Oates, Andrew C; Heisenberg, Carl-Philip; Brand, Michael

    2006-04-01

    Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

  13. A Model of Generating Visual Place Cells Based on Environment Perception and Similar Measure

    Directory of Open Access Journals (Sweden)

    Yang Zhou

    2016-01-01

    Full Text Available It is an important content to generate visual place cells (VPCs in the field of bioinspired navigation. By analyzing the firing characteristic of biological place cells and the existing methods for generating VPCs, a model of generating visual place cells based on environment perception and similar measure is abstracted in this paper. VPCs’ generation process is divided into three phases, including environment perception, similar measure, and recruiting of a new place cell. According to this process, a specific method for generating VPCs is presented. External reference landmarks are obtained based on local invariant characteristics of image and a similar measure function is designed based on Euclidean distance and Gaussian function. Simulation validates the proposed method is available. The firing characteristic of the generated VPCs is similar to that of biological place cells, and VPCs’ firing fields can be adjusted flexibly by changing the adjustment factor of firing field (AFFF and firing rate’s threshold (FRT.

  14. The role of starburst amacrine cells in visual signal processing

    Science.gov (United States)

    TAYLOR, W.R.; SMITH, R.G.

    2012-01-01

    Starburst amacrine cells (SBACs) within the adult mammalian retina provide the critical inhibition that underlies the receptive field properties of direction-selective ganglion cells (DSGCs). The SBACs generate direction-selective output of GABA that differentially inhibits the DSGCs. We review the biophysical mechanisms that produce directional GABA release from SBACs and test a network model that predicts the effects of reciprocal inhibition between adjacent SBACs. The results of the model simulations suggest that reciprocal inhibitory connections between closely spaced SBACs should be spatially selective, while connections between more widely spaced cells could be indiscriminate. SBACs were initially identified as cholinergic neurons and were subsequently shown to contain release both acetylcholine and GABA. While the role of the GABAergic transmission is well established, the role of the cholinergic transmission remains unclear. PMID:22310373

  15. [Endothelial origin for hematopoietic stem cells: a visual proof].

    Science.gov (United States)

    Boisset, Jean-Charles; Robin, Catherine

    2011-10-01

    Hematopoietic stem cells (HSC) are the source of all blood cell types produced during the entire life of an organism. They appear during embryonic development, where they will transit through different successive hematopoietic organs, before to finally colonize the bone marrow. Nowadays, the precise origin of HSC remains a matter of controversy. Different HSC precursor candidates, located in different anatomical sites, have been proposed. Here, we summarize and discuss the different theories in light of the recent articles, especially those using in vivo confocal microscopy technology.

  16. Visualizing how cancer chromosome abnormalities form in living cells

    Science.gov (United States)

    For the first time, scientists have directly observed events that lead to the formation of a chromosome abnormality that is often found in cancer cells. The abnormality, called a translocation, occurs when part of a chromosome breaks off and becomes attac

  17. 3D visualization of membrane failures in fuel cells

    Science.gov (United States)

    Singh, Yadvinder; Orfino, Francesco P.; Dutta, Monica; Kjeang, Erik

    2017-03-01

    Durability issues in fuel cells, due to chemical and mechanical degradation, are potential impediments in their commercialization. Hydrogen leak development across degraded fuel cell membranes is deemed a lifetime-limiting failure mode and potential safety issue that requires thorough characterization for devising effective mitigation strategies. The scope and depth of failure analysis has, however, been limited by the 2D nature of conventional imaging. In the present work, X-ray computed tomography is introduced as a novel, non-destructive technique for 3D failure analysis. Its capability to acquire true 3D images of membrane damage is demonstrated for the very first time. This approach has enabled unique and in-depth analysis resulting in novel findings regarding the membrane degradation mechanism; these are: significant, exclusive membrane fracture development independent of catalyst layers, localized thinning at crack sites, and demonstration of the critical impact of cracks on fuel cell durability. Evidence of crack initiation within the membrane is demonstrated, and a possible new failure mode different from typical mechanical crack development is identified. X-ray computed tomography is hereby established as a breakthrough approach for comprehensive 3D characterization and reliable failure analysis of fuel cell membranes, and could readily be extended to electrolyzers and flow batteries having similar structure.

  18. Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions.

    Directory of Open Access Journals (Sweden)

    Matthew L Katz

    Full Text Available Sensory stimuli are encoded by diverse kinds of neurons but the identities of the recorded neurons that are studied are often unknown. We explored in detail the firing patterns of eight previously defined genetically-identified retinal ganglion cell (RGC types from a single transgenic mouse line. We first introduce a new technique of deriving receptive field vectors (RFVs which utilises a modified form of mutual information ("Quadratic Mutual Information". We analysed the firing patterns of RGCs during presentation of short duration (~10 second complex visual scenes (natural movies. We probed the high dimensional space formed by the visual input for a much smaller dimensional subspace of RFVs that give the most information about the response of each cell. The new technique is very efficient and fast and the derivation of novel types of RFVs formed by the natural scene visual input was possible even with limited numbers of spikes per cell. This approach enabled us to estimate the 'visual memory' of each cell type and the corresponding receptive field area by calculating Mutual Information as a function of the number of frames and radius. Finally, we made predictions of biologically relevant functions based on the RFVs of each cell type. RGC class analysis was complemented with results for the cells' response to simple visual input in the form of black and white spot stimulation, and their classification on several key physiological metrics. Thus RFVs lead to predictions of biological roles based on limited data and facilitate analysis of sensory-evoked spiking data from defined cell types.

  19. Representation of the visual field in the primary visual area of the marmoset monkey: magnification factors, point-image size, and proportionality to retinal ganglion cell density.

    Science.gov (United States)

    Chaplin, Tristan A; Yu, Hsin-Hao; Rosa, Marcello G P

    2013-04-01

    The primary visual area (V1) forms a systematic map of the visual field, in which adjacent cell clusters represent adjacent points of visual space. A precise quantification of this map is key to understanding the anatomical relationships between neurons located in different stations of the visual pathway, as well as the neural bases of visual performance in different regions of the visual field. We used computational methods to quantify the visual topography of V1 in the marmoset (Callithrix jacchus), a small diurnal monkey. The receptive fields of neurons throughout V1 were mapped in two anesthetized animals using electrophysiological recordings. Following histological reconstruction, precise 3D reconstructions of the V1 surface and recording sites were generated. We found that the areal magnification factor (M(A) ) decreases with eccentricity following a function that has the same slope as that observed in larger diurnal primates, including macaque, squirrel, and capuchin monkeys, and humans. However, there was no systematic relationship between M(A) and polar angle. Despite individual variation in the shape of V1, the relationship between M(A) and eccentricity was preserved across cases. Comparison between V1 and the retinal ganglion cell density demonstrated preferential magnification of central space in the cortex. The size of the cortical compartment activated by a punctiform stimulus decreased from the foveal representation towards the peripheral representation. Nonetheless, the relationship between the receptive field sizes of V1 cells and the density of ganglion cells suggested that each V1 cell receives information from a similar number of retinal neurons, throughout the visual field.

  20. NaviCell Web Service for network-based data visualization.

    Science.gov (United States)

    Bonnet, Eric; Viara, Eric; Kuperstein, Inna; Calzone, Laurence; Cohen, David P A; Barillot, Emmanuel; Zinovyev, Andrei

    2015-07-01

    Data visualization is an essential element of biological research, required for obtaining insights and formulating new hypotheses on mechanisms of health and disease. NaviCell Web Service is a tool for network-based visualization of 'omics' data which implements several data visual representation methods and utilities for combining them together. NaviCell Web Service uses Google Maps and semantic zooming to browse large biological network maps, represented in various formats, together with different types of the molecular data mapped on top of them. For achieving this, the tool provides standard heatmaps, barplots and glyphs as well as the novel map staining technique for grasping large-scale trends in numerical values (such as whole transcriptome) projected onto a pathway map. The web service provides a server mode, which allows automating visualization tasks and retrieving data from maps via RESTful (standard HTTP) calls. Bindings to different programming languages are provided (Python and R). We illustrate the purpose of the tool with several case studies using pathway maps created by different research groups, in which data visualization provides new insights into molecular mechanisms involved in systemic diseases such as cancer and neurodegenerative diseases.

  1. Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda.

    Directory of Open Access Journals (Sweden)

    Laura E Valverde-Islas

    Full Text Available BACKGROUND: Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. METHODOLOGY/PRINCIPAL FINDINGS: Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. CONCLUSIONS/SIGNIFICANCE: We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

  2. Visualizing tropoelastin in a long-term human elastic fibre cell culture model.

    Science.gov (United States)

    Halm, M; Schenke-Layland, K; Jaspers, S; Wenck, H; Fischer, F

    2016-02-04

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin-fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models.

  3. Visualizing tropoelastin in a long-term human elastic fibre cell culture model

    Science.gov (United States)

    Halm, M.; Schenke-Layland, K.; Jaspers, S.; Wenck, H.; Fischer, F.

    2016-01-01

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models. PMID:26842906

  4. Globally visualizing the microtubule-dependent transport behaviors of influenza virus in live cells.

    Science.gov (United States)

    Liu, Shu-Lin; Zhang, Li-Juan; Wang, Zhi-Gang; Zhang, Zhi-Ling; Wu, Qiu-Mei; Sun, En-Ze; Shi, Yun-Bo; Pang, Dai-Wen

    2014-04-15

    Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.

  5. Real-time visualization of nanoparticles interacting with glioblastoma stem cells.

    Science.gov (United States)

    Pohlmann, Elliot S; Patel, Kaya; Guo, Sujuan; Dukes, Madeline J; Sheng, Zhi; Kelly, Deborah F

    2015-04-08

    Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics.

  6. Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

    Directory of Open Access Journals (Sweden)

    Angela RI Meyrelles

    2016-02-01

    Full Text Available This study investigated the rate of human papillomavirus (HPV persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample, re-infection (detection of different types of HPV in the 2 samples, and type-specific HPV persistence (the same HPV type found in both samples. An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30% or re-infection (20%. A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79. Our findings show that bonafide (type-specific HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

  7. Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

    Science.gov (United States)

    Meyrelles, Angela RI; Siqueira, Juliana D; dos Santos, Pâmela P; Hofer, Cristina B; Luiz, Ronir R; Seuánez, Héctor N; Almeida, Gutemberg; Soares, Marcelo A; Soares, Esmeralda A; Machado, Elizabeth S

    2016-01-01

    This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting. PMID:26872340

  8. Visualization and cellular hierarchy inference of single-cell data using SPADE.

    Science.gov (United States)

    Anchang, Benedict; Hart, Tom D P; Bendall, Sean C; Qiu, Peng; Bjornson, Zach; Linderman, Michael; Nolan, Garry P; Plevritis, Sylvia K

    2016-07-01

    High-throughput single-cell technologies provide an unprecedented view into cellular heterogeneity, yet they pose new challenges in data analysis and interpretation. In this protocol, we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. It was initially developed for flow and mass cytometry single-cell data. We describe SPADE's implementation and application using an open-source R package that runs on Mac OS X, Linux and Windows systems. A typical SPADE analysis on a 2.27-GHz processor laptop takes ∼5 min. We demonstrate the applicability of SPADE to single-cell RNA-seq data. We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm. We contrast the implementation and outputs of these methods for normal and malignant hematopoietic cells analyzed by mass cytometry and provide recommendations for appropriate use. Finally, we provide an integrative strategy that combines the strengths of t-SNE and SPADE to infer cellular hierarchy from high-dimensional single-cell data.

  9. Visualizing spatiotemporal dynamics of multicellular cell-cycle progressions with fucci technology.

    Science.gov (United States)

    Sakaue-Sawano, Asako; Miyawaki, Atsushi

    2014-05-01

    The visualization of cell-cycle behavior of individual cells within complex tissues presents an irresistible challenge to biologists studying multicellular structures. However, the transition from G1 to S in the cell cycle is difficult to monitor despite the fact that the process involves the critical decision to initiate a new round of DNA replication. Here, we use ubiquitination oscillators that control cell-cycle transitions to develop genetically encoded fluorescent probes for cell-cycle progression. Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes exploit the regulation of cell-cycle-dependent ubiquitination to effectively label individual nuclei in G1 phase red, and those in S/G2/M phases green. Cultured cells and transgenic mice constitutively expressing the probes have been generated, such that every cell nucleus shows either red or green fluorescence. This protocol details two experiments that use biological samples expressing Fucci probes. One experiment involves time-lapse imaging of cells stably expressing a Fucci derivative (Fucci2), which allows for the exploration of the spatiotemporal patterns of cell-cycle dynamics during structural and behavioral changes of cultured cells. The other experiment involves large-field, high-resolution imaging of fixed sections of Fucci transgenic mouse embryos, which provides maps that illustrate cell proliferation versus differentiation in various developing organs.

  10. Effects of deuterium oxide and galvanic vestibular stimulation on visual cortical cell function.

    Science.gov (United States)

    Reinis, S; Landolt, J P; Weiss, D S; Money, K E

    1984-03-01

    /he spontaneous and evoked unit activities of complex visual cortical cells were recorded from Brodmann's area 18 in immobilized, unanesthetized cats before, during, and after stimulation of the vestibular system. The vestibular system was stimulated by intravenous injection of deuterium oxide (D2O)--a noted nystagmogenic agent (14)--or by direct galvanic stimulation of the labyrinth. Measures of the receptive-field areas, poststimulus time histograms, directional preferences, and the optimal speed of the light bar stimulating the cell were obtained before and after the application of D2O. Directional preferences were determined in a novel manner, using a method derived from a hierarchical clustering technique (19). Data were collected and analyzed from a) visual cortical cells in cats with intact labyrinths, b) visual cortical cells in cats following bilateral labrinthectomies, and c) nonvisual cortical cells in cats with intact labyrinths. In cats with intact labyrinths, D2O changed the optimal length of the light bar that was able to stimulate the cortical cell as well as the path on which it evoked the response of the cell. Both values, which constitute the receptive field of the cell, changed approximately proportionately. This effect usually lasts for less than 4.5 h. The other cellular characteristics were also altered by the D2O. Galvanic stimulation of the labyrinth resembles, in its effects, the injection of D2O. In labyrinth-intact cats, the time course of area 18 spontaneous activity dramatically increased 30 min or more after D2O was administered. It peaked 2-3 h later and still had not returned to preinjection levels even 7 h after the D2O administration. In bilaterally labyrinthectomized cats, the spontaneous activity of the visual cells (and the other cellular characteristics studied) did not change following D2O administration. In nonvisual cells from labyrinth-intact cats, the spontaneous activity demonstrated a slight but significant decrease

  11. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

    Directory of Open Access Journals (Sweden)

    Alex Costa

    Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

  12. In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy

    Science.gov (United States)

    Egawa, Mariko; Tokunaga, Kyoya; Hosoi, Junichi; Iwanaga, Shinya; Ozeki, Yasuyuki

    2016-08-01

    Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

  13. Machine-Part cell formation through visual decipherable clustering of Self Organizing Map

    CERN Document Server

    Chattopadhyay, Manojit; Dan, Pranab K; 10.1007/s00170-010-2802-4

    2011-01-01

    Machine-part cell formation is used in cellular manufacturing in order to process a large variety, quality, lower work in process levels, reducing manufacturing lead-time and customer response time while retaining flexibility for new products. This paper presents a new and novel approach for obtaining machine cells and part families. In the cellular manufacturing the fundamental problem is the formation of part families and machine cells. The present paper deals with the Self Organising Map (SOM) method an unsupervised learning algorithm in Artificial Intelligence, and has been used as a visually decipherable clustering tool of machine-part cell formation. The objective of the paper is to cluster the binary machine-part matrix through visually decipherable cluster of SOM color-coding and labelling via the SOM map nodes in such a way that the part families are processed in that machine cells. The Umatrix, component plane, principal component projection, scatter plot and histogram of SOM have been reported in t...

  14. A cell-type-specific role for murine Commd1 in liver inflammation

    NARCIS (Netherlands)

    Bartuzi, Paulina; Wijshake, Tobias; Dekker, Daphne C.; Fedoseienko, Alina; Kloosterhuis, Niels J.; Youssef, Sameh A.; Li, Haiying; Shiri-Sverdlov, Ronit; Kuivenhoven, Jan-Albert; de Bruin, Alain; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bait

    2014-01-01

    The transcription factor NF-kappa B plays a critical role in the inflammatory response and it has been implicated in various diseases, including non-alcoholic fatty liver disease (NAFLD). Although transient NF-kappa B activation may protect tissues from stress, a prolonged NF-kappa B activation can

  15. eFORGE : A Tool for Identifying Cell Type-Specific Signal in Epigenomic Data

    NARCIS (Netherlands)

    Breeze, Charles E.; Paul, Dirk S.; van Dongen, Jenny; Butcher, Lee M.; Ambrose, John C.; Barrett, James E.; Lowe, Robert; Rakyan, Vardhman K.; Iotchkova, Valentina; Frontini, Mattia; Downes, Kate; Ouwehand, Willem H.; Laperle, Jonathan; Jacques, Pierre-ETienne; Bourque, Guillaume; Bergmann, Anke K.; Siebert, Reiner; Vellenga, Edo; Saeed, Sadia; Matarese, Filomena; Martens, Joost H. A.; Stunnenberg, Hendrik G.; Teschendorff, Andrew E.; Herrero, Javier; Birney, Ewan; Dunham, Ian; Beck, Stephan

    2016-01-01

    Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new stand-alone and

  16. Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific

    NARCIS (Netherlands)

    Jansen, Anne H P; van Hal, Maurik; Op den Kelder, Ilse C; Meier, Romy T; de Ruiter, Anna-Aster; Schut, Menno H; Smith, Donna L; Grit, Corien; Brouwer, Nieske; Kamphuis, Willem; Boddeke, H W G M; den Dunnen, Wilfred F A; van Roon, Willeke M C; Bates, Gillian P; Hol, Elly M; Reits, Eric A

    2017-01-01

    Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions cou

  17. Partitioning Heritability of Regulatory and Cell-Type-Specific Variants across 11 Common Diseases

    DEFF Research Database (Denmark)

    Gusev, Alexander; Lee, S Hong; Trynka, Gosia

    2014-01-01

    Regulatory and coding variants are known to be enriched with associations identified by genome-wide association studies (GWASs) of complex disease, but their contributions to trait heritability are currently unknown. We applied variance-component methods to imputed genotype data for 11 common...... enrichment. We replicated these findings but found no significant contribution from rare coding variants in independent schizophrenia cohorts genotyped on GWAS and exome chips. Our results highlight the value of analyzing components of heritability to unravel the functional architecture of common disease....

  18. Cell type-specific properties and environment shape tissue specificity of cancer genes.

    Science.gov (United States)

    Schaefer, Martin H; Serrano, Luis

    2016-02-09

    One of the biggest mysteries in cancer research remains why mutations in certain genes cause cancer only at specific sites in the human body. The poor correlation between the expression level of a cancer gene and the tissues in which it causes malignant transformations raises the question of which factors determine the tissue-specific effects of a mutation. Here, we explore why some cancer genes are associated only with few different cancer types (i.e., are specific), while others are found mutated in a large number of different types of cancer (i.e., are general). We do so by contrasting cellular functions of specific-cancer genes with those of general ones to identify properties that determine where in the body a gene mutation is causing malignant transformations. We identified different groups of cancer genes that did not behave as expected (i.e., DNA repair genes being tissue specific, immune response genes showing a bimodal specificity function or strong association of generally expressed genes to particular cancers). Analysis of these three groups demonstrates the importance of environmental impact for understanding why certain cancer genes are only involved in the development of some cancer types but are rarely found mutated in other types of cancer.

  19. Cell type specificity of tissue plasminogen activator in the mouse barrel cortex

    Directory of Open Access Journals (Sweden)

    Philip Chu

    2015-09-01

    Full Text Available We provide data in this article related to (C.C. Chen et al.,. Neurosci. Lett., 599 (2015 152–157. [1] where the expression of tissue plasminogen activator (tPA is expressed by the whisker representation in the somatosensory cortex. Here, we provide immunocytochemistry data indicating that tPA is expressed by putative excitatory neurons as well as parvalbumin+ interneurons but not by somatostatin+ inhibitory interneurons. We also provide data showing that microglia do not normally express high levels of tPA, but upregulate their levels following cortical penetration with a recording electrode.

  20. Input- and Cell-Type-Specific Endocannabinoid-Dependent LTD in the Striatum

    Directory of Open Access Journals (Sweden)

    Yu-Wei Wu

    2015-01-01

    Full Text Available Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1 receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.

  1. Cell type-specific delivery of short interfering RNAs by dye-functionalised theranostic nanoparticles

    Science.gov (United States)

    Press, Adrian T.; Traeger, Anja; Pietsch, Christian; Mosig, Alexander; Wagner, Michael; Clemens, Mark G.; Jbeily, Nayla; Koch, Nicole; Gottschaldt, Michael; Bézière, Nicolas; Ermolayev, Volodymyr; Ntziachristos, Vasilis; Popp, Jürgen; Kessels, Michael M.; Qualmann, Britta; Schubert, Ulrich S.; Bauer, Michael

    2014-12-01

    Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.

  2. Sample to answer visualization pipeline for low-cost point-of-care blood cell counting

    Science.gov (United States)

    Smith, Suzanne; Naidoo, Thegaran; Davies, Emlyn; Fourie, Louis; Nxumalo, Zandile; Swart, Hein; Marais, Philip; Land, Kevin; Roux, Pieter

    2015-03-01

    We present a visualization pipeline from sample to answer for point-of-care blood cell counting applications. Effective and low-cost point-of-care medical diagnostic tests provide developing countries and rural communities with accessible healthcare solutions [1], and can be particularly beneficial for blood cell count tests, which are often the starting point in the process of diagnosing a patient [2]. The initial focus of this work is on total white and red blood cell counts, using a microfluidic cartridge [3] for sample processing. Analysis of the processed samples has been implemented by means of two main optical visualization systems developed in-house: 1) a fluidic operation analysis system using high speed video data to determine volumes, mixing efficiency and flow rates, and 2) a microscopy analysis system to investigate homogeneity and concentration of blood cells. Fluidic parameters were derived from the optical flow [4] as well as color-based segmentation of the different fluids using a hue-saturation-value (HSV) color space. Cell count estimates were obtained using automated microscopy analysis and were compared to a widely accepted manual method for cell counting using a hemocytometer [5]. The results using the first iteration microfluidic device [3] showed that the most simple - and thus low-cost - approach for microfluidic component implementation was not adequate as compared to techniques based on manual cell counting principles. An improved microfluidic design has been developed to incorporate enhanced mixing and metering components, which together with this work provides the foundation on which to successfully implement automated, rapid and low-cost blood cell counting tests.

  3. A two- and three-dimensional approach for visualizing human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Brøchner, Christian Beltoft; Vestentoft, Peter S; Lynnerup, Niels;

    2010-01-01

    visualization of this 2D-expression pattern can be created by developing a 3D-model of the culture, based on serial paraffin sections. Individual sections are stained using individual markers. Using 3D image processing software such as Mimics or 3D-Doctor, the actual 3D-rendering of an entire colony can...... be accomplished. An extended version of this technique even allows for a high-magnification 3D-reconstruction of an area of interest (AOI), e.g., the developing hepatic stem cells. These techniques allow both a 2D and a 3D visualization of hESC colonies and lead to new insights into and information about...

  4. A visual targeting system for the microinjection of unstained adherent cells.

    Science.gov (United States)

    Becattini, Gabriele; Mattos, Leonardo S; Caldwell, Darwin G

    2013-02-01

    Automatic localization and targeting are critical steps in automating the process of microinjecting adherent cells. This process is currently performed manually by highly trained operators and is characterized as a laborious task with low success rate. Therefore, automation is desired to increase the efficiency and consistency of the operations. This research offers a contribution to this procedure through the development of a vision system for a robotic microinjection setup. Its goals are to automatically locate adherent cells in a culture dish and target them for a microinjection. Here the major concern was the achievement of an error-free targeting system to guarantee high consistency in microinjection experiments. To accomplish this, a novel visual targeting algorithm integrating different image processing techniques was proposed. This framework employed defocusing microscopy to highlight cell features and improve cell segmentation and targeting reliability. Three main image processing techniques, operating at three different focus levels in a bright field (BF) microscope, were used: an anisotropic contour completion (ACC) method, a local intensity variation background-foreground classifier, and a grayscale threshold-based segmentation. The proposed framework combined information gathered by each of these methods using a validation map and this was shown to provide reliable cell targeting results. Experiments conducted with sets of real images from two different cell lines (CHO-K1 and HEK), which contained a total of more than 650 cells, yielded flawless targeting results along with a cell detection ratio greater than 50%.

  5. [Understanding of immune system by visualization of spatiotemporal regulation of immune cells in the entire body].

    Science.gov (United States)

    Tomura, Michio

    2013-01-01

    Immune system is high-dimensional integrated system distributed in the whole body. Many kinds of, total 10(11) of immune cells are regulated by receiving appropriate signals in appropriate places. We have been attempting to understand immune system by revealing spatiotemporal regulation of immune cells at the whole body level by "Visualization of immune response in vivo". Photoconvertible protein, "Kaede"-Tg mice allowed us to monitor cell-replacement and cell-movement in the whole body by marking cells with color of Kaede from green to red with exposure to violet light. It is applicable to small cell number populations in both lymphoid organs and also peripheral tissues under both normal and pathophysiological conditions. By using this system, we have demonstrated novel findings that "Naive CD4(+) T cell recirculation is an active process that they recirculate through lymphoid organs to seek limited niche for interacting with endogenous antigens and upregulate their function." and "Activated regulatory T cells emigrating from cutaneous immune response is responsible for termination of immune reponse." I will introduce these new tools of us and would like to discuss what is needed to understand immune system in the entire body.

  6. VISUAL PERCEPTION BASED AUTOMATIC RECOGNITION OF CELL MOSAICS IN HUMAN CORNEAL ENDOTHELIUMMICROSCOPY IMAGES

    Directory of Open Access Journals (Sweden)

    Yann Gavet

    2011-05-01

    Full Text Available The human corneal endothelium can be observed with two types of microscopes: classical optical microscope for ex-vivo imaging, and specular optical microscope for in-vivo imaging. The quality of the cornea is correlated to the endothelial cell density and morphometry. Automatic methods to analyze the human corneal endothelium images are still not totally efficient. Image analysis methods that focus only on cell contours do not give good results in presence of noise and of bad conditions of acquisition. More elaborated methods introduce regional informations in order to performthe cell contours completion, thus implementing the duality contour-region. Their good performance can be explained by their connections with several basic principles of human visual perception (Gestalt Theory and Marr's computational theory.

  7. Alkyne-tag Raman imaging for visualization of mobile small molecules in live cells.

    Science.gov (United States)

    Yamakoshi, Hiroyuki; Dodo, Kosuke; Palonpon, Almar; Ando, Jun; Fujita, Katsumasa; Kawata, Satoshi; Sodeoka, Mikiko

    2012-12-26

    Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2'-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated.

  8. Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaida, Atsushi; Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp

    2013-10-04

    Highlights: •We visualized radiation-induced cell kinetics in spheroids. •HeLa-Fucci cells were used for detection of cell-cycle changes. •Radiation-induced G2 arrest was prolonged in the spheroid. •The inner and outer cell fractions behaved differently. -- Abstract: In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.

  9. Visualization of mitochondrial DNA replication in individual cells by EdU signal amplification.

    Science.gov (United States)

    Haines, Kristine M; Feldman, Eva L; Lentz, Stephen I

    2010-11-15

    Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

  10. Focal electrical stimulation of major ganglion cell types in the primate retina for the design of visual prostheses.

    Science.gov (United States)

    Jepson, Lauren H; Hottowy, Pawel; Mathieson, Keith; Gunning, Deborah E; Dabrowski, Wladyslaw; Litke, Alan M; Chichilnisky, E J

    2013-04-24

    Electrical stimulation of retinal neurons with an advanced retinal prosthesis may eventually provide high-resolution artificial vision to the blind. However, the success of future prostheses depends on the ability to activate the major parallel visual pathways of the human visual system. Electrical stimulation of the five numerically dominant retinal ganglion cell types was investigated by simultaneous stimulation and recording in isolated peripheral primate (Macaca sp.) retina using multi-electrode arrays. ON and OFF midget, ON and OFF parasol, and small bistratified ganglion cells could all be activated directly to fire a single spike with submillisecond latency using brief pulses of current within established safety limits. Thresholds for electrical stimulation were similar in all five cell types. In many cases, a single cell could be specifically activated without activating neighboring cells of the same type or other types. These findings support the feasibility of direct electrical stimulation of the major visual pathways at or near their native spatial and temporal resolution.

  11. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat

    2010-12-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  12. Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy.

    Science.gov (United States)

    Reyes, Nicholas L; Banks, Glen B; Tsang, Mark; Margineantu, Daciana; Gu, Haiwei; Djukovic, Danijel; Chan, Jacky; Torres, Michelle; Liggitt, H Denny; Hirenallur-S, Dinesh K; Hockenbery, David M; Raftery, Daniel; Iritani, Brian M

    2015-01-13

    Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I "red" slow twitch and type II "white" fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.

  13. Spatial phase sensitivity of complex cells in primary visual cortex depends on stimulus contrast.

    Science.gov (United States)

    Meffin, H; Hietanen, M A; Cloherty, S L; Ibbotson, M R

    2015-12-01

    Neurons in primary visual cortex are classified as simple, which are phase sensitive, or complex, which are significantly less phase sensitive. Previously, we have used drifting gratings to show that the phase sensitivity of complex cells increases at low contrast and after contrast adaptation while that of simple cells remains the same at all contrasts (Cloherty SL, Ibbotson MR. J Neurophysiol 113: 434-444, 2015; Crowder NA, van Kleef J, Dreher B, Ibbotson MR. J Neurophysiol 98: 1155-1166, 2007; van Kleef JP, Cloherty SL, Ibbotson MR. J Physiol 588: 3457-3470, 2010). However, drifting gratings confound the influence of spatial and temporal summation, so here we have stimulated complex cells with gratings that are spatially stationary but continuously reverse the polarity of the contrast over time (contrast-reversing gratings). By varying the spatial phase and contrast of the gratings we aimed to establish whether the contrast-dependent phase sensitivity of complex cells results from changes in spatial or temporal processing or both. We found that most of the increase in phase sensitivity at low contrasts could be attributed to changes in the spatial phase sensitivities of complex cells. However, at low contrasts the complex cells did not develop the spatiotemporal response characteristics of simple cells, in which paired response peaks occur 180° out of phase in time and space. Complex cells that increased their spatial phase sensitivity at low contrasts were significantly overrepresented in the supragranular layers of cortex. We conclude that complex cells in supragranular layers of cat cortex have dynamic spatial summation properties and that the mechanisms underlying complex cell receptive fields differ between cortical layers.

  14. Human Papilloma Virus prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy

    Directory of Open Access Journals (Sweden)

    Lloveras Belén

    2010-06-01

    Full Text Available Abstract Background Cervical cancer represents an important global public health problem. It is the 2nd most common cancer among women worldwide. Human Papillomavirus (HPV infection is now well-established as a necessary cause of invasive cervical cancer (ICC development. Only a few studies on HPV prevalence and type-specific distribution in ICC have been conducted in Italy. Aim To describe the prevalence of HPV and the HPV type-specific distribution in ICC cases identified in Rome, Italy. Methods 140 paraffin embedded tissue blocks of primary ICC diagnosed between 2001 and 2006 were identified at the Regina Elena Cancer Institute in Rome (Italy. HPV was detected through amplification of HPV DNA using SPF-10 HPV broad-spectrum primers followed by DEIA and then genotyping by LiPA25 (version 1. Results 134 cases were considered suitable for HPV DNA detection after histological evaluation; and overall, 90.3% (121/134 HPV prevalence was detected. 111 cases had a single HPV type, 4 cases had an uncharacterized type (HPVX and 6 cases had multiple HPV infections. The five most common single HPV types among positive cases were: HPV16 (71/121; 58.7%, HPV18 (12/121; 9.9%, HPV31, HPV45 and HPV58 (5/121; 4.1% each. 2 (1.5% of the single infections and 2 (1.5% of the multiple infections contained low risk types. Statistically significant differences in the relative contribution of HPV18 were found when comparing squamous cell carcinomas with adenocarcinomas. Conclusions HPV16 and HPV18 accounted for almost 70% of all the HPV positive ICC cases. The study provides baseline information for further evaluation on the impact of recently introduced HPV vaccines in Italy.

  15. Cell exclusion in couette flow: evaluation through flow visualization and mechanical forces.

    Science.gov (United States)

    Leslie, Laura J; Marshall, Lindsay J; Devitt, Andrew; Hilton, Andrew; Tansley, Geoff D

    2013-03-01

    Cell exclusion is the phenomenon whereby the hematocrit and viscosity of blood decrease in areas of high stress. While this is well known in naturally occurring Poiseuille flow in the human body, it has never previously been shown in Couette flow, which occurs in implantable devices including blood pumps. The high-shear stresses that occur in the gap between the boundaries in Couette flow are known to cause hemolysis in erythrocytes. We propose to mitigate this damage by initiating cell exclusion through the use of a spiral-groove bearing (SGB) that will provide escape routes by which the cells may separate themselves from the plasma and the high stresses in the gap. The force between two bearings (one being the SGB) in Couette flow was measured. Stained erythrocytes, along with silver spheres of similar diameter to erythrocytes, were visualized across a transparent SGB at various gap heights. A reduction in the force across the bearing for human blood, compared with fluids of comparable viscosity, was found. This indicates a reduction in the viscosity of the fluid across the bearing due to a lowered hematocrit because of cell exclusion. The corresponding images clearly show both cells and spheres being excluded from the gap by entering the grooves. This is the first time the phenomenon of cell exclusion has been shown in Couette flow. It not only furthers our understanding of how blood responds to different flows but could also lead to improvements in the future design of medical devices.

  16. Visualization of sialic acid produced on bacterial cell surfaces by lectin staining.

    Science.gov (United States)

    Kajiwara, Hitomi; Toda, Munetoyo; Mine, Toshiki; Nakada, Hiroshi; Wariishi, Hiroyuki; Yamamoto, Takeshi

    2010-01-01

    Oligosaccharides containing N-acetylneuraminic acid on the cell surface of some pathogenic bacteria are important for host-microbe interactions. N-acetylneuraminic acid (Neu5Ac) plays a major role in the pathogenicity of bacterial pathogens. For example, cell surface sialyloligosaccharide moieties of the human pathogen Haemophilus influenzae are involved in virulence and adhesion to host cells. In this study, we have established a method of visualizing Neu5Ac linked to a glycoconjugate on the bacterial cell surface based on lectin staining. Photobacterium damselae strain JT0160, known to produce a-2,6-sialyltransferase, was revealed to possess Neu5Ac by HPLC. Using the strain, a strong Sambucus sieboldiana lectin-binding signal was detected. The bacteria producing α-2,6-sialyltransferases could be divided into two groups: those with a lot of α-2,6-linked Neu5Ac on the cell surface and those with a little. In the present study, we developed a useful method for evaluating the relationship between Neu5Ac expression on the cell surface and the degree of virulence of marine bacteria.

  17. Visualizing the Histotripsy Process: Bubble Cloud-Cancer Cell Interactions in a Tissue-Mimicking Environment.

    Science.gov (United States)

    Vlaisavljevich, Eli; Maxwell, Adam; Mancia, Lauren; Johnsen, Eric; Cain, Charles; Xu, Zhen

    2016-10-01

    Histotripsy is a non-invasive ultrasonic ablation method that uses cavitation to mechanically fractionate tissue into acellular debris. With a sufficient number of pulses, histotripsy can completely fractionate tissue into a liquid-appearing homogenate with no cellular structures. The location, shape and size of lesion formation closely match those of the cavitation cloud. Previous work has led to the hypothesis that the rapid expansion and collapse of histotripsy bubbles fractionate tissue by inducing large stress and strain on the tissue structures immediately adjacent to the bubbles. In the work described here, the histotripsy bulk tissue fractionation process is visualized at the cellular level for the first time using a custom-built 2-MHz transducer incorporated into a microscope stage. A layer of breast cancer cells were cultured within an optically transparent fibrin-based gel phantom to mimic cells inside a 3-D extracellular matrix. To test the hypothesis, the cellular response to single and multiple histotripsy pulses was investigated using high-speed optical imaging. Bubbles were always generated in the extracellular space, and significant cell displacement/deformation was observed for cells directly adjacent to the bubble during both bubble expansion and collapse. The largest displacements were observed during collapse for cells immediately adjacent to the bubble, with cells moving more than 150-300 μm in less than 100 μs. Cells often underwent multiple large deformations (>150% strain) over multiple pulses, resulting in the bisection of cells multiple times before complete removal. To provide theoretical support to the experimental observations, a numerical simulation was conducted using a single-bubble model, which indicated that histotripsy exerts the largest strains and cell displacements in the regions immediately adjacent to the bubble. The experimental and simulation results support our hypothesis, which helps to explain the formation of the

  18. Visualizing the endocytosis of phenylephrine in living cells by quantum dot-based tracking.

    Science.gov (United States)

    Ma, Jing; Wu, Lina; Hou, Zhun; Song, Yao; Wang, Lei; Jiang, Wei

    2014-08-01

    To study the intracellular receptor-drug transportation, a fluorescent probe consisting of phenylephrine-polyethylene glycol-quantum dots conjugate was employed to track endocytosis process of phenylephrine in living cells. This type of movement was studied by continuously filming fluorescent images in the same cell. We also calculated the movement parameters, and divided the endocytosis process into 6 stages. Furthermore, the movement parameters of this probe in different organelles were determined by co-localization of the probe fluorescent images and different cellular organelles. After comparing the parameters in cellular organelles with these in 6 stages, the whole endocytosis pathway was demonstrated. These results verified that this probe successfully tracked the whole intracellular dynamic endocytosis process of phenylephrine. Our method realized the visual tracking the whole receptor-mediated endocytosis, which is a new approach on investigating the molecular mechanisms and kinetic properties of intracellular receptor-drug transportation.

  19. Pyramidal cells make specific connections onto smooth (GABAergic neurons in mouse visual cortex.

    Directory of Open Access Journals (Sweden)

    Rita Bopp

    2014-08-01

    Full Text Available One of the hallmarks of neocortical circuits is the predominance of recurrent excitation between pyramidal neurons, which is balanced by recurrent inhibition from smooth GABAergic neurons. It has been previously described that in layer 2/3 of primary visual cortex (V1 of cat and monkey, pyramidal cells filled with horseradish peroxidase connect approximately in proportion to the spiny (excitatory, 95% and 81%, respectively and smooth (GABAergic, 5% and 19%, respectively dendrites found in the neuropil. By contrast, a recent ultrastructural study of V1 in a single mouse found that smooth neurons formed 51% of the targets of the superficial layer pyramidal cells. This suggests that either the neuropil of this particular mouse V1 had a dramatically different composition to that of V1 in cat and monkey, or that smooth neurons were specifically targeted by the pyramidal cells in that mouse. We tested these hypotheses by examining similar cells filled with biocytin in a sample of five mice. We found that the average composition of the neuropil in V1 of these mice was similar to that described for cat and monkey V1, but that the superficial layer pyramidal cells do form proportionately more synapses with smooth dendrites than the equivalent neurons in cat or monkey. These distributions may underlie the distinct differences in functional architecture of V1 between rodent and higher mammals.

  20. Focal Electrical Stimulation of Major Ganglion Cell Types in the Primate Retina for the Design of Visual Prostheses

    OpenAIRE

    2013-01-01

    Electrical stimulation of retinal neurons with an advanced retinal prosthesis may eventually provide high-resolution artificial vision to the blind. However, the success of future prostheses depends on the ability to activate the major parallel visual pathways of the human visual system. Electrical stimulation of the five numerically dominant retinal ganglion cell types was investigated by simultaneous stimulation and recording in isolated peripheral primate (Macaca sp.) retina using multi-el...

  1. Human papillomavirus type-specific prevalence in the cervical cancer screening population of Czech women.

    Directory of Open Access Journals (Sweden)

    Ruth Tachezy

    Full Text Available BACKGROUND: Infection with high-risk human papillomavirus (HPVtypes has been recognized as a causal factor for the development of cervical cancer and a number of other malignancies. Today, vaccines against HPV, highly effective in the prevention of persistent infection and precancerous lesions, are available for the routine clinical practice. OBJECTIVES: The data on the prevalence and type-specific HPV distribution in the population of each country are crucial for the surveillance of HPV type-specific prevalence at the onset of vaccination against HPV. METHODS: Women attending a preventive gynecological examination who had no history of abnormal cytological finding and/or surgery for cervical lesions were enrolled. All samples were tested for the presence of HPV by High-Risk Hybrid Capture 2 (HR HC2 and by a modified PCR-reverse line blot assay with broad spectrum primers (BS-RLB. RESULTS: Cervical smears of 1393 women were analyzed. In 6.5% of women, atypical cytological findings were detected. Altogether, 28.3% (394/1393 of women were positive for any HPV type by BS-RLB, 18.2% (254/1393 by HR HC2, and 22.3% (310/1393 by BS-RLB for HR HPV types. In women with atypical findings the prevalence for HR and any HPV types were significantly higher than in women with normal cytological findings. Overall, 36 different HPV types were detected, with HPV 16 being the most prevalent (4.8%. HPV positivity decreased with age; the highest prevalence was 31.5% in the age group 21-25 years. CONCLUSIONS: Our study subjects represent the real screening population. HPV prevalence in this population in the Czech Republic is higher than in other countries of Eastern Europe. Also the spectrum of the most prevalent HPV types differs from those reported by others but HPV 16 is, concordantly, the most prevalent type. Country-specific HPV type-specific prevalences provide baseline information which will enable to measure the impact of HPV vaccination in the future.

  2. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    Science.gov (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  3. Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology

    Science.gov (United States)

    Martinez, Victor D.; Vucic, Emily A.; Thu, Kelsie L.; Hubaux, Roland; Enfield, Katey S.S.; Pikor, Larissa A.; Becker-Santos, Daiana D.; Brown, Carolyn J.; Lam, Stephen; Lam, Wan L.

    2015-01-01

    Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to “piRNA clusters” reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology. PMID:26013764

  4. Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells.

    Science.gov (United States)

    Yamazaki, Daiki; Yoshida, Shigeo; Asami, Tadao; Kuchitsu, Kazuyuki

    2003-07-01

    Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

  5. Fibre type-specific change in FXYD1 phosphorylation during acute intense exercise in humans

    DEFF Research Database (Denmark)

    Thomassen, Martin; Murphy, Robyn M; Bangsbo, Jens

    2013-01-01

    The aim of the present study was to examine fibre type-specific Na(+)-K(+) pump subunit expression and exercise-induced alterations in phospholemman (FXYD1) phosphorylation in humans. Segments of human skeletal muscle fibres were dissected and fibre typed, and protein expression was determined...... by Western blotting. The protein expression of the Na(+)-K(+) pump a2 isoform was lower in type I than in type II fibres (0.63 ± 0.04 a.u. vs. 1.00 ± 0.07 a.u., P ... channel Kir6.2 was higher in type I compared with type II fibres. In both type I (P type II fibres (P

  6. Muscle fiber-type distribution, fiber-type-specific damage, and the Pompe disease phenotype.

    Science.gov (United States)

    van den Berg, L E M; Drost, M R; Schaart, G; de Laat, J; van Doorn, P A; van der Ploeg, A T; Reuser, A J J

    2013-09-01

    Pompe disease is a lysosomal storage disorder caused by acid α-glucosidase deficiency and characterized by progressive muscle weakness. Enzyme replacement therapy (ERT) has ameliorated patients' perspectives, but reversal of skeletal muscle pathology remains a challenge. We studied pretreatment biopsies of 22 patients with different phenotypes to investigate to what extent fiber-type distribution and fiber-type-specific damage contribute to clinical diversity. Pompe patients have the same fiber-type distribution as healthy persons, but among nonclassic patients with the same GAA mutation (c.-32-13T>G), those with early onset of symptoms tend to have more type 2 muscle fibers than those with late-onset disease. Further, it seemed that the older, more severely affected classic infantile patients and the wheelchair-bound and ventilated nonclassic patients had a greater proportion of type 2x muscle fibers. However, as in other diseases, this may be caused by physical inactivity of those patients.

  7. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    Science.gov (United States)

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  8. Turtle Dorsal Cortex Pyramidal Neurons Comprise Two Distinct Cell Types with Indistinguishable Visual Responses.

    Directory of Open Access Journals (Sweden)

    Thomas Crockett

    Full Text Available A detailed inventory of the constituent pieces in cerebral cortex is considered essential to understand the principles underlying cortical signal processing. Specifically, the search for pyramidal neuron subtypes is partly motivated by the hypothesis that a subtype-specific division of labor could create a rich substrate for computation. On the other hand, the extreme integration of individual neurons into the collective cortical circuit promotes the hypothesis that cellular individuality represents a smaller computational role within the context of the larger network. These competing hypotheses raise the important question to what extent the computational function of a neuron is determined by its individual type or by its circuit connections. We created electrophysiological profiles from pyramidal neurons within the sole cellular layer of turtle visual cortex by measuring responses to current injection using whole-cell recordings. A blind clustering algorithm applied to these data revealed the presence of two principle types of pyramidal neurons. Brief diffuse light flashes triggered membrane potential fluctuations in those same cortical neurons. The apparently network driven variability of the visual responses concealed the existence of subtypes. In conclusion, our results support the notion that the importance of diverse intrinsic physiological properties is minimized when neurons are embedded in a synaptic recurrent network.

  9. Retinal ganglion cell projections to the hamster suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: bifurcation and melanopsin immunoreactivity

    Science.gov (United States)

    Morin, Lawrence P.; Blanchard, Jane H.; Provencio, Ignacio

    2003-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) receives direct retinal input via the retinohypothalamic tract (RHT), and the retinal ganglion cells contributing to this projection may be specialized with respect to direct regulation of the circadian clock. However, some ganglion cells forming the RHT bifurcate, sending axon collaterals to the intergeniculate leaflet (IGL) through which light has secondary access to the circadian clock. The present studies provide a more extensive examination of ganglion cell bifurcation and evaluate whether ganglion cells projecting to several subcortical visual nuclei contain melanopsin, a putative ganglion cell photopigment. The results showed that retinal ganglion cells projecting to the SCN send collaterals to the IGL, olivary pretectal nucleus, and superior colliculus, among other places. Melanopsin-immunoreactive (IR) ganglion cells are present in the hamster retina, and some of these cells project to the SCN, IGL, olivary pretectal nucleus, or superior colliculus. Triple-label analysis showed that melanopsin-IR cells bifurcate and project bilaterally to each SCN, but not to the other visual nuclei evaluated. The melanopsin-IR cells have photoreceptive characteristics optimal for circadian rhythm regulation. However, the presence of moderately widespread bifurcation among ganglion cells projecting to the SCN, and projection by melanopsin-IR cells to locations distinct from the SCN and without known rhythm function, suggest that this ganglion cell type is generalized, rather than specialized, with respect to the conveyance of photic information to the brain. Copyright 2003 Wiley-Liss, Inc.

  10. Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro

    Science.gov (United States)

    Qi, Shuhong; Zhang, Zhihong

    2016-03-01

    Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.

  11. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

    Science.gov (United States)

    Laguerre, Aurélien; Wong, Judy M. Y.; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  12. Differences in Visual-Spatial Input May Underlie Different Compression Properties of Firing Fields for Grid Cell Modules in Medial Entorhinal Cortex.

    Science.gov (United States)

    Raudies, Florian; Hasselmo, Michael E

    2015-11-01

    Firing fields of grid cells in medial entorhinal cortex show compression or expansion after manipulations of the location of environmental barriers. This compression or expansion could be selective for individual grid cell modules with particular properties of spatial scaling. We present a model for differences in the response of modules to barrier location that arise from different mechanisms for the influence of visual features on the computation of location that drives grid cell firing patterns. These differences could arise from differences in the position of visual features within the visual field. When location was computed from the movement of visual features on the ground plane (optic flow) in the ventral visual field, this resulted in grid cell spatial firing that was not sensitive to barrier location in modules modeled with small spacing between grid cell firing fields. In contrast, when location was computed from static visual features on walls of barriers, i.e. in the more dorsal visual field, this resulted in grid cell spatial firing that compressed or expanded based on the barrier locations in modules modeled with large spacing between grid cell firing fields. This indicates that different grid cell modules might have differential properties for computing location based on visual cues, or the spatial radius of sensitivity to visual cues might differ between modules.

  13. Use of fluorescent proteins and color-coded imaging to visualize cancer cells with different genetic properties.

    Science.gov (United States)

    Hoffman, Robert M

    2016-03-01

    Fluorescent proteins are very bright and available in spectrally-distinct colors, enable the imaging of color-coded cancer cells growing in vivo and therefore the distinction of cancer cells with different genetic properties. Non-invasive and intravital imaging of cancer cells with fluorescent proteins allows the visualization of distinct genetic variants of cancer cells down to the cellular level in vivo. Cancer cells with increased or decreased ability to metastasize can be distinguished in vivo. Gene exchange in vivo which enables low metastatic cancer cells to convert to high metastatic can be color-coded imaged in vivo. Cancer stem-like and non-stem cells can be distinguished in vivo by color-coded imaging. These properties also demonstrate the vast superiority of imaging cancer cells in vivo with fluorescent proteins over photon counting of luciferase-labeled cancer cells.

  14. Three counting methods agree on cell and neuron number in chimpanzee primary visual cortex

    Directory of Open Access Journals (Sweden)

    Daniel James Miller

    2014-05-01

    Full Text Available Determining the cellular composition of specific brain regions is crucial to our understanding of the function of neurobiological systems. It is therefore useful to identify the extent to which different methods agree when estimating the same properties of brain circuitry. In this study, we estimated the number of neuronal and non-neuronal cells in the primary visual cortex (area 17 or V1 of both hemispheres from a single chimpanzee. Specifically, we processed samples distributed across V1 of the right hemisphere after cortex was flattened into a sheet using two variations of the isotropic fractionator cell and neuron counting method. We processed the left hemisphere as serial brain slices for stereological investigation. The goal of this study was to evaluate the agreement between these methods in the most direct manner possible by comparing estimates of cell density across one brain region of interest in a single individual. In our hands, these methods produced similar estimates of the total cellular population (approximately 1 billion as well as the number of neurons (approximately 675 million in chimpanzee V1, providing evidence that both techniques estimate the same parameters of interest. In addition, our results indicate the strengths of each distinct tissue preparation procedure, highlighting the importance of attention to anatomical detail. In summary, we found that the isotropic fractionator and the stereological optical fractionator produced concordant estimates of the cellular composition of V1, and that this result supports the conclusion that chimpanzees conform to the primate pattern of exceptionally high packing density in V1. Ultimately, our data suggest that investigators can optimize their experimental approach by using any of these counting methods to obtain reliable cell and neuron counts.

  15. Human muscle fiber type-specific insulin signaling: impact of obesity and type 2 diabetes.

    Science.gov (United States)

    Albers, Peter H; Pedersen, Andreas J T; Birk, Jesper B; Kristensen, Dorte E; Vind, Birgitte F; Baba, Otto; Nøhr, Jane; Højlund, Kurt; Wojtaszewski, Jørgen F P

    2015-02-01

    Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber type-specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1α (PDH-E1α) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1α). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.

  16. Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

    Science.gov (United States)

    Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

    2015-11-15

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and

  17. Visualization of probiotic-mediated Ca2+ signaling in intestinal epithelial cells in vivo

    Directory of Open Access Journals (Sweden)

    Takahiro Adachi

    2016-12-01

    Full Text Available Probiotics, such as lactic acid bacteria (LAB and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs, because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60 transgenic mouse line and established 5D (x, y, z, time, and Ca2+ intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed Bacillus subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions, and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

  18. Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo

    Science.gov (United States)

    Adachi, Takahiro; Kakuta, Shigeru; Aihara, Yoshiko; Kamiya, Tomonori; Watanabe, Yohei; Osakabe, Naomi; Hazato, Naoki; Miyawaki, Atsushi; Yoshikawa, Soichiro; Usami, Takako; Karasuyama, Hajime; Kimoto-Nira, Hiromi; Hirayama, Kazuhiro; Tsuji, Noriko M.

    2016-01-01

    Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria. PMID:28018362

  19. Comparative study on direction selectivity and functional organization of the primary visual cortical cells in monkeys and cats

    Institute of Scientific and Technical Information of China (English)

    寿天德; 周逸峰; 俞洪波

    2000-01-01

    Although the directionally selective cells in many visual cortical areas are organized in columnar manner, the functional organization of direction selectivity of area VI in the monkey still remains unclear. We quantitatively studied the proportion of directionally selective cells, direction selectivity and the functional organization of the striate cortical cells in the monkey and compared those with the cat. The results show that the direction selectivity and directional organization of striate cortical cells in the monkey are significantly weaker than those in the cat, suggesting that the species difference between the two kinds of animal is related to their different anatomic pathways.

  20. Comparative study on direction selectivity and functional organization of the primary visual cortical cells in monkeys and cats

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Although the directionally selective cells in many visual cortical areas are organized in columnar manner, the functional organization of direction selectivity of area Vl in the monkey still remains unclear. We quantitatively studied the proportion of directionally selective cells, direction selectivity and the functional organization of the striate cortical cells in the monkey and compared those with the cat. The results show that the direction selectivity and directional organization of striate cortical cells in the monkey are significantly weaker than those in the cat, suggesting that the species difference between the two kinds of animal is related to their different anatomic pathways.

  1. [Persistence of orientation-selective cells of the primary visual cortex in kittens enucleated unilaterally at birth and reared in darkness].

    Science.gov (United States)

    Fregnac, Y; Buisseret, P; Bienenstock, E; Gary-Bobo, E; Imbert, M

    1978-07-17

    In kittens dark reared (6 weeks old) orientation selective cells are no longer recorded in the primary visual cortex, while in kittens of same age, enucleated at birth unilaterally and reared in identical conditions, 30% of visual cortical cells are shown to be orientation selective and in addition respond preferentially to horizontal and vertical orientations.

  2. Streptococcus pneumoniae Transmission Is Blocked by Type-Specific Immunity in an Infant Mouse Model

    Science.gov (United States)

    Zangari, Tonia; Wang, Yang

    2017-01-01

    ABSTRACT Epidemiological studies on Streptococcus pneumoniae show that rates of carriage are highest in early childhood and that the major benefit of the pneumococcal conjugate vaccine (PCV) is a reduction in the incidence of nasopharyngeal colonization through decreased transmission within a population. In this study, we sought to understand how anti-S. pneumoniae immunity affects nasal shedding of bacteria, the limiting step in experimental pneumococcal transmission. Using an infant mouse model, we examined the role of immunity (passed from mother to pup) on shedding and within-litter transmission of S. pneumoniae by pups infected at 4 days of life. Pups from both previously colonized immune and PCV-vaccinated mothers had higher levels of anti-S. pneumoniae IgG than pups from non-immune or non-vaccinated mothers and shed significantly fewer S. pneumoniae over the first 5 days of infection. By setting up cross-foster experiments, we demonstrated that maternal passage of antibody to pups either in utero or post-natally decreases S. pneumoniae shedding. Passive immunization experiments showed that type-specific antibody to capsular polysaccharide is sufficient to decrease shedding and that the agglutinating function of immunoglobulin is required for this effect. Finally, we established that anti-pneumococcal immunity and anti-PCV vaccination block host-to-host transmission of S. pneumoniae. Moreover, immunity in either the donor or recipient pups alone was sufficient to reduce rates of transmission, indicating that decreased shedding and protection from acquisition of colonization are both contributing factors. Our findings provide a mechanistic explanation for the reduced levels of S. pneumoniae transmission between hosts immune from prior exposure and among vaccinated children. PMID:28292980

  3. World Health Organization Guidelines for Containment of Poliovirus Following Type-Specific Polio Eradication - Worldwide, 2015.

    Science.gov (United States)

    Previsani, Nicoletta; Tangermann, Rudolph H; Tallis, Graham; Jafari, Hamid S

    2015-08-28

    In 1988, the World Health Assembly of the World Health Organization (WHO) resolved to eradicate polio worldwide. Among the three wild poliovirus (WPV) types (type 1, type 2, and type 3), WPV type 2 (WPV2) has been eliminated in the wild since 1999, and WPV type 3 (WPV3) has not been reported since 2012. In 2015, only Afghanistan and Pakistan have reported WPV transmission. On May 25, 2015, all WHO Member States endorsed World Health Assembly resolution 68.3 on full implementation of the Polio Eradication and Endgame Strategic Plan 2013-2018 (the Endgame Plan), and with it, the third Global Action Plan to minimize poliovirus facility-associated risk (GAPIII). All WHO Member States have committed to implementing appropriate containment of WPV2 in essential laboratory and vaccine production facilities* by the end of 2015 and of type 2 oral poliovirus vaccine (OPV2) within 3 months of global withdrawal of OPV2, which is planned for April 2016. This report summarizes critical steps for essential laboratory and vaccine production facilities that intend to retain materials confirmed to contain or potentially containing type-specific WPV, vaccine-derived poliovirus (VDPV), or OPV/Sabin viruses, and steps for nonessential facilities† that process specimens that contain or might contain polioviruses. National authorities will need to certify that the essential facilities they host meet the containment requirements described in GAPIII. After certification of WPV eradication, the use of all OPV will cease; final containment of all polioviruses after polio eradication and OPV cessation will minimize the risk for reintroduction of poliovirus into a polio-free world.

  4. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    Science.gov (United States)

    Eilers, Wouter; Gevers, Wouter; van Overbeek, Daniëlle; de Haan, Arnold; Jaspers, Richard T.; Hilbers, Peter A.; van Riel, Natal; Flück, Martin

    2014-01-01

    We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII) contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE) coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis) and slow-type muscle (soleus) for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02). In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII. PMID:25054156

  5. Muscle-Type Specific Autophosphorylation of CaMKII Isoforms after Paced Contractions

    Directory of Open Access Journals (Sweden)

    Wouter Eilers

    2014-01-01

    Full Text Available We explored to what extent isoforms of the regulator of excitation-contraction and excitation-transcription coupling, calcium/calmodulin protein kinase II (CaMKII contribute to the specificity of myocellular calcium sensing between muscle types and whether concentration transients in its autophosphorylation can be simulated. CaMKII autophosphorylation at Thr287 was assessed in three muscle compartments of the rat after slow or fast motor unit-type stimulation and was compared against a computational model (CaMuZclE coupling myocellular calcium dynamics with CaMKII Thr287 phosphorylation. Qualitative differences existed between fast- (gastrocnemius medialis and slow-type muscle (soleus for the expression pattern of CaMKII isoforms. Phospho-Thr287 content of δA CaMKII, associated with nuclear functions, demonstrated a transient and compartment-specific increase after excitation, which contrasted to the delayed autophosphorylation of the sarcoplasmic reticulum-associated βM CaMKII. In soleus muscle, excitation-induced δA CaMKII autophosphorylation demonstrated frequency dependence (P = 0.02. In the glycolytic compartment of gastrocnemius medialis, CaMKII autophosphorylation after excitation was blunted. In silico assessment emphasized the importance of mitochondrial calcium buffer capacity for excitation-induced CaMKII autophosphorylation but did not predict its isoform specificity. The findings expose that CaMKII autophosphorylation with paced contractions is regulated in an isoform and muscle type-specific fashion and highlight properties emerging for phenotype-specific regulation of CaMKII.

  6. Ultrastructural visualization of the Mesenchymal-to-Epithelial Transition during reprogramming of human fibroblasts to induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    M.K. Høffding

    2015-01-01

    Here, we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming, as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6–12 days after electroporation, in synchrony with the upregulation of early pluripotency markers, and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT which takes place during mammalian gastrulation.

  7. Linear Multi-Epitope (Glyco)peptides for Type-specific Serology of Herpes Simplex Virus (HSV) infections.

    Science.gov (United States)

    Risinger, Christian Walter; Sørensen, Kasper Kildegaard; Jensen, Knud J; Olofsson, Sigvard; Bergstrom, Tomas; Blixt, Ola

    2017-02-26

    Detection of type-specific antibodies is an important and essential part of accurate diagnosis, even in silent carriers of HSV-1 (oral) and HSV-2 (genital) infections. Serologic assays that identify HSV-1 and HSV-2 type-specific antibodies have been commercially available for more than a decade but often face problems related to cross-reactivity and similar issues. Attempts to identify type specific peptide epitopes for use in serology for both HSV-1 and HSV-2 have been limited. We recently demonstrated epitope mapping of envelope glycoprotein G2 and identified a type-specific glycopeptide epitope that broadly recognized HSV-2 infected individuals. In the present work we have performed a comprehensive glycopeptide synthesis and microarray epitope mapping of 14 envelope proteins from HSV-1 and HSV-2, namely: gB, gC, gD, gE, gG, gH and gI, using sera from HSV-1 and HSV-2 infected individuals and control sera. Several unique type-specific peptide epitopes with a high cumulative sensitivity were identified and synthesized as one large linear multi-epitope sequence using microwave assisted solid-phase-(glyco)peptide synthesis. Microarray validation with clinically defined HSV and Varicella Zoster (VZV) sera confirmed excellent specificities and sensitivities.

  8. In vivo visualizing the dynamics of bone marrow stem cells in mouse retina and choroidal-retinal circulation

    Science.gov (United States)

    Wang, Heuy-Ching H.; Zwick, Harry; Edsall, Peter R.; Cheramie, Rachel D.; Lund, David J.; Stuck, Bruce

    2007-02-01

    It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. Therefore it is an attractive therapeutic intervention to apply autologous bone marrow-derived stem cells that may offer neuroprotection to laser-induced retinal injuries. The purpose of this study is to develop a method with which to visualize bone marrow stem cells dynamics in mouse retinal circulation. We have used a physiological method, confocal scanning laser ophthalmoscope (SLO), to track the highly enriched stem/progenitor cells circulating in the retina. Stem cells were enriched by immunomagnetic depletion of cells committed to the T- and B lymphocytic, myeloid and erythorid lineages. CellTracker TM Green-labeled stem cells were injected into the tail veins of mice with laser-induced focal retinal injuries. Bone marrow stem cells labeled with CellTracker TM Green were visible in the retinal circulation for as long as 1 hour and 30 minutes. These studies suggest that stem cell-enriched bone marrow cells may have the ability to mobilize into laser-induced retinal injuries and possibly further proliferate, differentiate and functionally integrate into the retina.

  9. Kinetics of linear rouleaux formation studied by visual monitoring of red cell dynamic organization.

    Science.gov (United States)

    Barshtein, G; Wajnblum, D; Yedgar, S

    2000-05-01

    Red blood cells (RBCs) in the presence of plasma proteins or other macromolecules may form aggregates, normally in rouleaux formations, which are dispersed with increasing blood flow. Experimental observations have suggested that the spontaneous aggregation process involves the formation of linear rouleaux (FLR) followed by formation of branched rouleaux networks. Theoretical models for the spontaneous rouleaux formation were formulated, taking into consideration that FLR may involve both "polymerization," i.e., interaction between two single RBCs (e + e) and the addition of a single RBC to the end of an existing rouleau (e + r), as well as "condensation" between two rouleaux by end-to-end addition (r + r). The present study was undertaken to experimentally examine the theoretical models and their assumptions, by visual monitoring of the spontaneous FLR (from singly dispersed RBC) in plasma, in a narrow gap flow chamber. The results validate the theoretical model, showing that FLR involves both polymerization and condensation, and that the kinetic constants for the above three types of intercellular interactions are the same, i.e., k(ee) = k(er) = k(rr) = k, and for all tested hematocrits (0.625-6%) k < 0.13 +/- 0.03 s(-1).

  10. Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaofei [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036 (China); Deng, Ping; Cui, Hongguang [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada); Wang, Aiming, E-mail: aiming.wang@agr.gc.ca [Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3 (Canada)

    2015-11-15

    Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.

  11. Distinct representation and distribution of visual information by specific cell types in mouse superficial superior colliculus.

    Science.gov (United States)

    Gale, Samuel D; Murphy, Gabe J

    2014-10-01

    The superficial superior colliculus (sSC) occupies a critical node in the mammalian visual system; it is one of two major retinorecipient areas, receives visual cortical input, and innervates visual thalamocortical circuits. Nonetheless, the contribution of sSC neurons to downstream neural activity and visually guided behavior is unknown and frequently neglected. Here we identified the visual stimuli to which specific classes of sSC neurons respond, the downstream regions they target, and transgenic mice enabling class-specific manipulations. One class responds to small, slowly moving stimuli and projects exclusively to lateral posterior thalamus; another, comprising GABAergic neurons, responds to the sudden appearance or rapid movement of large stimuli and projects to multiple areas, including the lateral geniculate nucleus. A third class exhibits direction-selective responses and targets deeper SC layers. Together, our results show how specific sSC neurons represent and distribute diverse information and enable direct tests of their functional role.

  12. Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond.

    Science.gov (United States)

    Liu, Kuang-Kai; Chen, Mei-Fang; Chen, Po-Yi; Lee, Tony J F; Cheng, Chia-Liang; Chang, Chia-Ching; Ho, Yen-Peng; Chao, Jui-I

    2008-05-21

    Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

  13. Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters.

    Directory of Open Access Journals (Sweden)

    Sunil Laxman

    Full Text Available BACKGROUND: During continuous growth in specific chemostat cultures, budding yeast undergo robust oscillations in oxygen consumption that are accompanied by highly periodic changes in transcript abundance of a majority of genes, in a phenomenon called the Yeast Metabolic Cycle (YMC. This study uses fluorescent reporters of genes specific to different YMC phases in order to visualize this phenomenon and understand the temporal regulation of gene expression at the level of individual cells within the cycling population. METHODOLOGY: Fluorescent gene expression reporters for different phases of the YMC were constructed and stably integrated into the yeast genome. Subsequently, these reporter-expressing yeast were used to visualize YMC dynamics at the individual cell level in cultures grown in a chemostat or in a microfluidics platform under varying glucose concentrations, using fluorescence microscopy and quantitative Western blots. CONCLUSIONS: The behavior of single cells within a metabolic cycling population was visualized using phase-specific fluorescent reporters. The reporters largely recapitulated genome-specified mRNA expression profiles. A significant fraction of the cell population appeared to exhibit basal expression of the reporters, supporting the hypothesis that there are at least two distinct subpopulations of cells within the cycling population. Although approximately half of the cycling population initiated cell division in each permissive window of the YMC, metabolic synchrony of the population was maintained. Using a microfluidics platform we observed that low glucose concentrations appear to be necessary for metabolic cycling. Lastly, we propose that there is a temporal window in the oxidative growth phase of the YMC where the cycling population segregates into at least two subpopulations, one which will enter the cell cycle and one which does not.

  14. Toehold-initiated rolling circle amplification for visualizing individual microRNAs in situ in single cells.

    Science.gov (United States)

    Deng, Ruijie; Tang, Longhua; Tian, Qianqian; Wang, Ying; Lin, Lei; Li, Jinghong

    2014-02-24

    The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA-mediated regulatory circuits and their disease associations. A toehold-initiated strand-displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold-initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.

  15. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data.

    Directory of Open Access Journals (Sweden)

    Damian J Matuszewski

    Full Text Available Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell measurements to population statistics. The software imports measurements from a simple text file, visualizes population distributions in a compact and comprehensive way, and can create gates for subpopulation classes based on control samples. We validate the tool by showing how PopulationProfiler can be used to analyze the effect of drugs that disturb the cell cycle, and compare the results to those obtained with flow cytometry.

  16. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data.

    Science.gov (United States)

    Matuszewski, Damian J; Wählby, Carolina; Puigvert, Jordi Carreras; Sintorn, Ida-Maria

    2016-01-01

    Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s) for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell measurements to population statistics. The software imports measurements from a simple text file, visualizes population distributions in a compact and comprehensive way, and can create gates for subpopulation classes based on control samples. We validate the tool by showing how PopulationProfiler can be used to analyze the effect of drugs that disturb the cell cycle, and compare the results to those obtained with flow cytometry.

  17. In-situ visualization of N2 evolution in operating direct hydrazine hydrate fuel cell by soft X-ray radiography

    Science.gov (United States)

    Sakamoto, Tomokazu; Deevanhxay, Phengxay; Asazawa, Koichiro; Tsushima, Shohji; Hirai, Shuichiro; Tanaka, Hirohisa

    2014-04-01

    Soft X-ray radiography technique was firstly applied to operating direct hydrazine hydrate fuel cell (DHFCs) in order to visualize N2 gas behaviors with high spatial and temporal resolution. Two different cells for in-situ visualization of N2 gas in the DHFCs in in-plane and through-plane direction were designed and fabricated. The utilization of soft X-ray made the visualization of generated N2 behavior in the DHFC possible with the spatial resolution of 1.5 μm and the temporal resolution of 2.0 s frame-1. In the in-plane visualization, the inhomogeneous N2 gas distribution, suggesting non-uniform reaction distribution in the anode of DHFC, was observed. In the through-plane visualization, N2 gas accumulation under the rib of anode and discharge to the channel was clearly observed, which are related with cell performance instability.

  18. Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

    Science.gov (United States)

    Nanton, Minelva R; Lee, Seung-Joo; Atif, Shaikh M; Nuccio, Sean-Paul; Taylor, Justin J; Bäumler, Andreas J; Way, Sing Sing; McSorley, Stephen J

    2015-02-01

    CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

  19. Visualization and quantitative analysis of extrachromosomal telomere-repeat DNA in individual human cells by Halo-FISH.

    Science.gov (United States)

    Komosa, Martin; Root, Heather; Meyn, M Stephen

    2015-02-27

    Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain 300), range widely in length (200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.

  20. Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

    1998-03-01

    Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

  1. Visualization of molecular composition and functionality of cancer cells using nanoparticle-augmented ultrasound-guided photoacoustics

    Science.gov (United States)

    Mallidi, Srivalleesha; Kim, Seungsoo; Karpiouk, Andrei; Joshi, Pratixa P.; Sokolov, Konstantin; Emelianov, Stanislav

    2015-01-01

    Assessment of molecular signatures of tumors in addition to their anatomy and morphology is desired for effective diagnostic and therapeutic procedures. Development of in vivo imaging techniques that can identify and monitor molecular composition of tumors remains an important challenge in pre-clinical research and medical practice. Here we present a molecular photoacoustic imaging technique that can visualize the presence and activity of an important cancer biomarker – epidermal growth factor receptor (EGFR), utilizing the effect of plasmon resonance coupling between molecular targeted gold nanoparticles. Specifically, spectral analysis of photoacoustic images revealed profound changes in the optical absorption of systemically delivered EGFR-targeted gold nanospheres due to their molecular interactions with tumor cells overexpressing EGFR. In contrast, no changes in optical properties and, therefore, photoacoustic signal, were observed after systemic delivery of non-targeted gold nanoparticles to the tumors. The results indicate that multi-wavelength photoacoustic imaging augmented with molecularly targeted gold nanoparticles has the ability to monitor molecular specific interactions between nanoparticles and cell-surface receptors, allowing visualization of the presence and functional activity of tumor cells. Furthermore, the approach can be used for other cancer cell-surface receptors such as human epidermal growth factor receptor 2 (HER2). Therefore, ultrasound-guided molecular photoacoustic imaging can potentially aid in tumor diagnosis, selection of customized patient-specific treatment, and monitor the therapeutic progression and outcome in vivo. PMID:25893171

  2. Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis.

    Science.gov (United States)

    Shiba, Tomonori; Mii, Masahiro

    2005-12-01

    Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.

  3. Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and in vivo visualization by magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Spray David C

    2011-02-01

    Full Text Available Abstract Background Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs have been used to label and visualize various cell types with magnetic resonance imaging (MRI. In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs labeled with clinically approved SPIONs. Results Rat and mouse MSCs were isolated, cultured, and incubated with dextran-covered SPIONs (ferumoxide alone or with poly-L-lysine (PLL or protamine chlorhydrate for 4 or 24 hrs. Labeling efficiency was evaluated by dextran immunocytochemistry and MRI. Cell proliferation and viability were evaluated in vitro with Ki67 immunocytochemistry and live/dead assays. Ferumoxide-labeled MSCs could be induced to differentiate to adipocytes, osteocytes and chondrocytes. We analyzed ferumoxide retention in MSCs with or without mitomycin C pretreatment. Approximately 95% MSCs were labeled when incubated with ferumoxide for 4 or 24 hrs in the presence of PLL or protamine, whereas labeling of MSCs incubated with ferumoxide alone was poor. Proliferative capacity was maintained in MSCs incubated with ferumoxide and PLL for 4 hrs, however, after 24 hrs it was reduced. MSCs incubated with ferumoxide and protamine were efficiently visualized by MRI; they maintained proliferation and viability for up to 7 days and remained competent to differentiate. After 21 days MSCs pretreated with mitomycin C still showed a large number of ferumoxide-labeled cells. Conclusions The efficient and long lasting uptake and retention of SPIONs by MSCs using a protocol

  4. Three dimensional visualization and fractal analysis of mosaic patches in rat chimeras: cell assortment in liver, adrenal cortex and cornea.

    Science.gov (United States)

    Iannaccone, Stephen; Zhou, Yue; Walterhouse, David; Taborn, Greg; Landini, Gabriel; Iannaccone, Philip

    2012-01-01

    The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations.

  5. Three dimensional visualization and fractal analysis of mosaic patches in rat chimeras: cell assortment in liver, adrenal cortex and cornea.

    Directory of Open Access Journals (Sweden)

    Stephen Iannaccone

    Full Text Available The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations.

  6. A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

    Institute of Scientific and Technical Information of China (English)

    Min QING; Zhi-ming YUAN; Pei-Yong Shi

    2009-01-01

    Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting flavivirus type.Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (ⅰ) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; (ⅱ)the neutralized VLPs are used to infect Vero cells; and (ⅲ) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT; importantly, it shortens the assay time from >10 days to <1 day, and can be performed in biosafety level-2 facility.

  7. Human Embryonic and Hepatic Stem Cell Differentiation Visualized in Two and Three Dimensions Based on Serial Sections

    DEFF Research Database (Denmark)

    Vestentoft, Peter S.; Brøchner, Christian B; Lynnerup, Niels;

    2015-01-01

    ESC colony, and prepare 3-5 μm thick serial sections. Immunohistochemistry applied to individual sections produces a 2-dimensional survey of the developing hESC colony. Based on serial paraffin sections of the 2D-expression pattern, a new and useful 3D-visualization can be modeled. The actual 3D rendering......Pluripotent human embryonic stem cells (hESCs) are characterized by two defining properties, self-renewal and differentiation. Self-renewing hESCs express transcription factors OCT4, SOX2, and NANOG, and surface markers SSEA-4 and TRA-1-60 and TRA-1-81 and their ability to differentiate...... into derivatives of the three germ layers show the differentiating potential. Studies suggest a certain microheterogeneity of the hESC colonies, in which not all cells in one colony of apparently undifferentiated cells express all the expected markers. We describe a technique to paraffin embed an entire h...

  8. CDy6, a photostable probe for long-term real-time visualization of mitosis and proliferating cells.

    Science.gov (United States)

    Jeong, Yun-Mi; Duanting, Zhai; Hennig, Holger; Samanta, Animesh; Agrawalla, Bikram Keshari; Bray, Mark-Anthony; Carpenter, Anne E; Chang, Young-Tae

    2015-02-19

    Long-term real-time visualization of lysosomal dynamics has been challenging at the onset of mitosis due to the lack of fluorescent probes enabling convenient imaging of dividing cells. We developed a long-term real-time photostable mitotic or proliferating marker, CDy6, a BODIPY-derived compound of designation yellow 6, which labels lysosome. In long-term real-time, CDy6 displayed a sharp increase in intensity and change in localization in mitosis, improved photostability, and decreased toxicity compared with other widely used lysosomal and DNA markers, and the ability to label cells in mouse xenograft models. Therefore, CDy6 may open new possibilities to target and trace lysosomal contents during mitosis and to monitor cell proliferation, which can further our knowledge of the basic underlying biological mechanisms in the management of cancer.

  9. Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus.

    Science.gov (United States)

    Liu, Haibin; Liu, Yi; Liu, Shulin; Pang, Dai-Wen; Xiao, Gengfu

    2011-07-01

    Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.

  10. Bioluminescence-based visualization of CD4 T cell dynamics using a T lineage-specific luciferase transgenic model1

    Directory of Open Access Journals (Sweden)

    Zinn Kurt R

    2009-08-01

    Full Text Available Abstract Background Rapid clonal expansion of T cells occurs in response to antigenic challenges. The kinetics of the T cell response has previously been described using tissue-based studies performed at defined time points. Luciferase bioluminescence has recently been utilized for non-invasive analysis of in vivo biologic processes in real-time. Results We have created a novel transgenic mouse model (T-Lux using a human CD2 mini-gene to direct luciferase expression specifically to the T cell compartment. T-Lux T cells demonstrated normal homing patterns within the intact mouse and following adoptive transfer. Bioluminescent signal correlated with T cell numbers in the whole body images as well as within specific organ regions of interest. Following transfer into lymphopenic (RAG2-/- recipients, homeostatic proliferation of T-Lux T cells was visualized using bioluminescent imaging. Real-time bioluminescent analysis of CD4+ T cell antigen-specific responses enabled real-time comparison of the kinetics and magnitude of clonal expansion and contraction in the inductive lymph node and tissue site of antigen injection. T cell expansion was dose-dependent despite the presence of supraphysiologic numbers of OVA-specific OT-II transgenic TCR T-Lux T cells. CD4+ T cells subsequently underwent a rapid (3–4 day contraction phase in the draining lymph node, with a delayed contraction in the antigen delivery site, with bioluminescent signal diminished below initial levels, representing TCR clonal frequency control. Conclusion The T-Lux mouse provides a novel, efficient model for tracking in vivo aspects of the CD4+ T cell response to antigen, providing an attractive approach for studies directed at immunotherapy or vaccine design.

  11. Cell surgery and growth factors in dry age-related macular degeneration: visual prognosis and morphological study

    Science.gov (United States)

    Limoli, Paolo Giuseppe; Limoli, Celeste; Vingolo, Enzo Maria; Scalinci, Sergio Zaccaria; Nebbioso, Marcella

    2016-01-01

    Background The aim of this research was to study the overall restoration effect on residual retinal cells through surgically grafted autologous cells onto the surrounding tissue, choroid and retina in order to produce a constant secretion of growth factors (GFs) in dry age-related macular degeneration (AMD) patients. Results 6 months after surgery, several values were statistically significant in the group with higher RTA. Also patient compliance analysis (PCA) in relation to functional change perception appeared to be very good. Methods Thirty-six eyes of 25 patients (range 64-84 years of age) affected by dry AMD were included in study, and divided in two groups by spectral domain-optical coherence tomography (SD-OCT): group A with retinal thickness average (RTA) less than 250 microns (μm) and group B with RTA equal to or more than 250 μm. Adipocytes, adipose-derived stem cells from the stromal-vascular fraction, and platelets from platelet-rich plasma were implanted in the suprachoroidal space. Particularly, the following parameters were evaluated: best corrected visual acuity (BCVA) for far and near distance, retinal thickness maps, scotopic and photopic electroretinogram (ERG), and microperimetry (MY). All statistical analyses were performed with STATA 14.0 (Collage Station, Texas, USA). Conclusions The available set of GFs allowed biological retinal neuroenhancement. After 6 months it improved visual performance (VP), but the increase was better if RTA recorded by OCT was higher, probably in relation to the presence of areas with greater cellularity. PMID:27391437

  12. Coding for stimulus velocity by temporal patterning of spike discharges in visual cells of cat superior colliculus.

    Science.gov (United States)

    Mandl, G

    1993-07-01

    Statistical analyses, performed on extracellularly recorded spike trains generated by 69 single motion sensitive visual cells in the intermediate layers of superior colliculi of pretrigeminal cat preparations, revealed that--even in the unstimulated condition (38/69)--most neuronal spike discharge patterns tended to switch between two stochastically distinct states, in the form of rapidly alternating "bursting" (high frequency) and "resting" (low frequency) episodes. The numbers of consecutive interspike intervals within a given state were, as a rule, independent integer-valued random variables with discrete probability distributions, in essential agreement with the semi-Markov model proposed by Ekholm and Hyvärinen [(1970) Biophysical Journal, 10, 773-796]. The introduction of visual stimuli (47/69) moving with velocities of 2-160 deg/sec caused systematic and reproducible changes in the ratio of bursting to resting activities, decreases in overall discharge variability, and increases in signal transinformation flow. Moreover, with one group of stimulated cells (28/47), increasing stimulus velocity caused increasingly precise ("stimulus-forced") synchronization of bursting episodes with specific phases of stimulus movement; while for a smaller group (12/47), stimulus-related alternations between bursting and resting states assumed the form of semi-rhythmical burst discharges within the characteristic 60-80 Hz "gamma oscillation" range ("stimulus-induced" synchronization). For a minority of cells (7/47), switching between bursting and resting states--although characteristically modified by stimulus velocity--remained largely desynchronized with all phases of stimulus transit. It was argued that such temporal patterns of discharge may constitute elements of a candidate "distribution" code for movement detection by the cat visual system.

  13. Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Crew, Jennifer R.; Falzari, Kanakeshwari [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States); DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu [Department of Cell Biology and Anatomy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064 (United States)

    2010-04-01

    Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed to differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.

  14. Adult stem cells in mice : visualization and characterization using genetic mouse models

    NARCIS (Netherlands)

    Snippert, H.J.G.

    2011-01-01

    The onset of each living organism starts with pluripotent stem cells that have the ability to differentiate into all the different cell types of an organism. However, during the earliest stages of development, the pluripotent stem cells will stepwise lose their developmental potential. The cells tha

  15. How a (subcellular coincidence detection mechanism featuring layer-5 pyramidal cells may help produce various visual phenomena

    Directory of Open Access Journals (Sweden)

    Talis eBachmann

    2015-12-01

    Full Text Available Perceptual phenomena such as spatio-temporal illusions and masking are typically explained by psychological (cognitive processing theories or large-scale neural theories involving inter-areal connectivity and neural circuits comprising of hundreds or more interconnected single cells. Subcellular mechanisms are hardly used for such purpose. Here a mechanistic theoretical view is presented on how a subcellular brain mechanism of integration of presynaptic signals that arrive at different compartments of layer-5 pyramidal neurons could explain a couple of spatiotemporal visual-phenomenal effects unfolding along very brief time intervals within the range of sub-second temporal scale.

  16. Visual Impairment

    Science.gov (United States)

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Visual Impairment KidsHealth > For Teens > Visual Impairment Print A ... with the brain, making vision impossible. What Is Visual Impairment? Many people have some type of visual ...

  17. Visual field

    Science.gov (United States)

    Perimetry; Tangent screen exam; Automated perimetry exam; Goldmann visual field exam; Humphrey visual field exam ... Confrontation visual field exam : This is a quick and basic check of the visual field. The health care provider ...

  18. Developmentally programmed 3' CpG island methylation confers tissue- and cell-type-specific transcriptional activation

    Science.gov (United States)

    During development, a small but significant number of CpG islands (CGIs) becomes methylated. The timing of developmentally programmed CGI methylation and associated mechanisms of transcriptional regulation during cellular differentiation, however, remain poorly characterized. Here we used genome-wid...

  19. Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific

    NARCIS (Netherlands)

    C.P.J. de Kock (Christiaan); B. Sakmann (Bert)

    2009-01-01

    textabstractSensation involves active movement of sensory organs, but it remains unknown how position or movement of sensory organs is encoded in cortex. In the rat whisker system, each whisker is represented by an individual cortical (barrel) column. Here, we quantified in awake, head-fixed rats th

  20. Opening up the blackbox: an interpretable deep neural network-based classifier for cell-type specific enhancer predictions

    OpenAIRE

    Kim, Seong Gon; Theera-Ampornpunt, Nawanol; Fang, Chih-Hao; Harwani, Mrudul; Grama, Ananth; Chaterji, Somali

    2016-01-01

    Background Gene expression is mediated by specialized cis-regulatory modules (CRMs), the most prominent of which are called enhancers. Early experiments indicated that enhancers located far from the gene promoters are often responsible for mediating gene transcription. Knowing their properties, regulatory activity, and genomic targets is crucial to the functional understanding of cellular events, ranging from cellular homeostasis to differentiation. Recent genome-wide investigation of epigeno...

  1. Comparative immunological characterization of type-specific and conserved B-cell epitopes of pinniped, felid and canid herpesviruses.

    NARCIS (Netherlands)

    M. Lebich; T.C. Harder (Timm); H.R. Frey; I.K.G. Visser (Ilona); A.D.M.E. Osterhaus (Albert); B. Liess

    1994-01-01

    textabstractMurine monoclonal antibodies (MAbs) were generated against phocid herpesviruses (PhHV 2557/Han88 and 7848/Han90) isolated from European harbour seals (Phoca vitulina), and against strains of both felid (FHV strain FVR 605) and canid herpesviruses (CHV isolate 5105/Han89). MAbs were chara

  2. The fiber-type specific response of skeletal muscle satellite cells to high-intensity resistance training in dialysis patients

    DEFF Research Database (Denmark)

    Molsted, Stig; Andersen, Jesper Løvind; Harrison, Adrian Paul;

    2015-01-01

    weekly. SC and myonuclear number were determined by immunohistochemistry of vastus lateralis muscle biopsy cross-sections. Knee extension torque was tested in a dynamometer. Results. During training SCs/type I fibers increased by 15%, whereas SCs/type II fibers remained unchanged. Myonuclear content...... of type II, but not type I, fibers increased with training. Before the control period, the SC content of type II fibers was lower than type I fibers, whereas contents were comparable when normalized to fiber area. Torque increased after training. Discussion. Increased myonuclear content of type II muscle...

  3. The intestinal epithelium during damage and regeneration : cell type-specific responses in experimental colitis and after cytostatic drug treatment

    NARCIS (Netherlands)

    I.B. Renes (Ingrid)

    2002-01-01

    textabstractIn the first part of this thesis the role of the colonic epithelium and in particular its associated mucus-layer during IBD and in several experimental colitis models is discussed (Chapter 2). In Chapter 3-5 our investigations regarding the colonic epithelium in rat during the different

  4. Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin.

    Science.gov (United States)

    Sano, Toshio; Higaki, Takumi; Oda, Yoshihisa; Hayashi, Tomomi; Hasezawa, Seiichiro

    2005-11-01

    The actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFP-fimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G2 phase and functions as a marker of cytokinesis.

  5. T Cell Receptor Activation of NF-κB in Effector T Cells: Visualizing Signaling Events Within and Beyond the Cytoplasmic Domain of the Immunological Synapse.

    Science.gov (United States)

    Traver, Maria K; Paul, Suman; Schaefer, Brian C

    2017-01-01

    The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells. Additionally, in effector T cells, much signaling occurs at cytoplasmic locations distant from the IS. Visualization of these cytoplasmic signaling complexes has provided key insights into the complex and dynamic regulation of NF-κB signal transduction in effector T cells. In this chapter, we provide in-depth protocols for activating and preparing effector T cells for fluorescence imaging, as well as a discussion of the effective application of distinct imaging methodologies, including confocal and super-resolution microscopy and imaging flow cytometry.

  6. Visualization-aided classification ensembles discriminate lung adenocarcinoma and squamous cell carcinoma samples using their gene expression profiles.

    Directory of Open Access Journals (Sweden)

    Ao Zhang

    Full Text Available INTRODUCTION: The widespread application of microarray experiments to cancer research is astounding including lung cancer, one of the most common fatal human tumors. Among non-small cell lung carcinoma (NSCLC, there are two major histological types of NSCLC, adenocarcinoma (AC and squamous cell carcinoma (SCC. RESULTS: In this paper, we proposed to integrate a visualization method called Radial Coordinate Visualization (Radviz with a suitable classifier, aiming at discriminating two NSCLC subtypes using patients' gene expression profiles. Our analyses on simulated data and a real microarray dataset show that combining with a classification method, Radviz may play a role in selecting relevant features and ameliorating parsimony, while the final model suffers no or least loss of accuracy. Most importantly, a graphic representation is more easily understandable and implementable for a clinician than statistical methods and/or mathematic equations. CONCLUSION: To conclude, using the NSCLC microarray data presented here as a benchmark, the comprehensive understanding of the underlying mechanism associated with NSCLC and of the mechanisms with its subtypes and respective stages will become reality in the near future.

  7. Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Ziomkiewicz, Iwona; Schulz, Alexander

    2013-01-01

    present the first super-resolution images of cellulose bundles in the plant cell wall produced by direct stochastic optical reconstruction microscopy (dSTORM) in combination with total internal reflection fluorescence (TIRF) microscopy. Since TIRF limits observation to the cell surface, we tested...... microscopy of some 200 nm in xy and 550 nm in z for green light, restricts the direct visualization of cellulose to relatively large bundles, whereas the structure of cellulose microfibrils with their diameter below 10 nm remains unresolved. Over the last decade, several so-called super-resolution microscopy...... confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. Conclusions Super-resolution light microscopy of PFS-stained cellulose fibrils is possible...

  8. Super-resolution imaging with Pontamine Fast Scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells

    DEFF Research Database (Denmark)

    Liesche, Johannes; Ziomkiewicz, Iwona; Schulz, Alexander

    2013-01-01

    of cellulose fibril orientation and growth. The fluorescent dye Pontamine Fast Scarlet 4BS (PFS) was shown to stain cellulose with high specificity and could be used to visualize cellulose bundles in cell walls of Arabidopsis root epidermal cells with confocal microscopy. The resolution limit of confocal...... as alternatives 3D-structured illumination microscopy (3D-SIM) and confocal microscopy, combined with image deconvolution. Both methods offer lower resolution than STORM, but enable 3D imaging. While 3D-SIM produced strong artifacts, deconvolution gave good results. The resolution was improved over conventional...... confocal microscopy and the approach could be used to demonstrate differences in fibril orientation in different layers of the cell wall as well as particular cellulose fortifications around plasmodesmata. Conclusions Super-resolution light microscopy of PFS-stained cellulose fibrils is possible...

  9. Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes.

    Science.gov (United States)

    Raulf, Alexandra; Voeltz, Nadine; Korzus, Daniel; Fleischmann, Bernd K; Hesse, Michael

    2017-02-24

    Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

  10. Prevalence of type-specific HPV infection by age and grade of cervical cytology: data from the ARTISTIC trial.

    Science.gov (United States)

    Sargent, A; Bailey, A; Almonte, M; Turner, A; Thomson, C; Peto, J; Desai, M; Mather, J; Moss, S; Roberts, C; Kitchener, H C

    2008-05-20

    Human papillomavirus (HPV) infection causes cervical cancer and premalignant dysplasia. Type-specific HPV prevalence data provide a basis for assessing the impact of HPV vaccination programmes on cervical cytology. We report high-risk HPV (HR-HPV) type-specific prevalence data in relation to cervical cytology for 24,510 women (age range: 20-64; mean age 40.2 years) recruited into the ARTISTIC trial, which is being conducted within the routine NHS Cervical Screening Programme in Greater Manchester. The most common HR-HPV types were HPV16, 18, 31, 51 and 52, which accounted for 60% of all HR-HPV types detected. There was a marked decline in the prevalence of HR-HPV infection with age, but the proportion due to each HPV type did not vary greatly with age. Multiple infections were common below the age of 30 years but less so between age 30 and 64 years. Catch-up vaccination of this sexually active cohort would be expected to reduce the number of women with moderate or worse cytology by 45%, but the number with borderline or mild cytology would fall by only 7%, giving an overall reduction of 12% in the number of women with abnormal cytology and 27% in the number with any HR-HPV infection. In the absence of broader cross-protection, the large majority of low-grade and many high-grade abnormalities may still occur in sexually active vaccinated women.

  11. Fiber type specific expression of TNF-alpha, IL-6 and IL-18 in human skeletal muscles.

    Science.gov (United States)

    Plomgaard, Peter; Penkowa, Milena; Pedersen, Bente K

    2005-01-01

    Skeletal muscle is now recognized as an endocrine organ with the capacity to produce signal peptides in response to muscle contractions. Here we demonstrate that resting healthy human muscles express cytokines in a fiber type specific manner. Human muscle biopsies from seven healthy young males were obtained from m. triceps, m. quadriceps vastus lateralis and m. soleus. Type I fibers contributed (mean +/- SE) 24.0 +/- 2.5% in triceps of total fibers, 51.3 +/- 2.4% in vastus and 84.9 +/- 22% in soleus. As expected, differences in the fiber type composition were accompanied by marked differences between the three muscles with regard to MHC I and MHC IIa mRNA expression. Immunohistochemistry demonstrated that tumor necrosis factor (TNF)-alpha and interleukin (IL)-18 were solely expressed by type II fibers, whereas the expression of IL-6 was more prominent in type I compared to type II fibers. The fiber type specificity was found in triceps, vastus and soleus indicating that the level of daily muscle activity did not influence basal cytokine expression. The specificity of cytokine expression in different muscle fiber types in healthy young males suggests that cytokines may play specific regulatory roles in normal physiology.

  12. Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

    Science.gov (United States)

    Ludmerer, S W; Benincasa, D; Mark, G E

    1996-01-01

    Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism. PMID:8676509

  13. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.

    Science.gov (United States)

    Wyckoff, Jeffrey B; Wang, Yarong; Lin, Elaine Y; Li, Jiu-feng; Goswami, Sumanta; Stanley, E Richard; Segall, Jeffrey E; Pollard, Jeffrey W; Condeelis, John

    2007-03-15

    Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors.

  14. Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere

    DEFF Research Database (Denmark)

    Steidle, A.; Sigl, K.; Schuhegger, R.

    2001-01-01

    -negative derivatives of Pseudomonas putida IsoF and Serratia liquefaciens MG1, two strains that are capable of colonizing tomato roots. These AHL monitor strains were used to visualize communication between defined bacterial populations in the rhizosphere of axenically grown tomato plants. Furthermore, we integrated...... into the chromosome of AHL-negative P. putida strain F117 an AHL sensor cassette that responds to the presence of long-chain AHLs with the expression of Gfp. This monitor strain was used to demonstrate that the indigenous bacterial community colonizing the roots of tomato plants growing in nonsterile soil produces...

  15. Visualization of immediate immune responses to pioneer metastatic cells in the lung.

    Science.gov (United States)

    Headley, Mark B; Bins, Adriaan; Nip, Alyssa; Roberts, Edward W; Looney, Mark R; Gerard, Audrey; Krummel, Matthew F

    2016-03-24

    Lung metastasis is the lethal determinant in many cancers and a number of lines of evidence point to monocytes and macrophages having key roles in its development. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue, and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed 'waves' of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer anti-metastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

  16. Visual bone marrow mesenchymal stem cell transplantation in the repair of spinal cord injury

    Directory of Open Access Journals (Sweden)

    Rui-ping Zhang

    2015-01-01

    Full Text Available An important factor in improving functional recovery from spinal cord injury using stem cells is maximizing the number of transplanted cells at the lesion site. Here, we established a contusion model of spinal cord injury by dropping a weight onto the spinal cord at T 7-8 . Superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells were transplanted into the injured spinal cord via the subarachnoid space. An outer magnetic field was used to successfully guide the labeled cells to the lesion site. Prussian blue staining showed that more bone marrow mesenchymal stem cells reached the lesion site in these rats than in those without magnetic guidance or superparamagnetic iron oxide labeling, and immunofluorescence revealed a greater number of complete axons at the lesion site. Moreover, the Basso, Beattie and Bresnahan (BBB locomotor rating scale scores were the highest in rats with superparamagnetic labeling and magnetic guidance. Our data confirm that superparamagnetic iron oxide nanoparticles effectively label bone marrow mesenchymal stem cells and impart sufficient magnetism to respond to the external magnetic field guides. More importantly, superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells can be dynamically and non-invasively tracked in vivo using magnetic resonance imaging. Superparamagnetic iron oxide labeling of bone marrow mesenchymal stem cells coupled with magnetic guidance offers a promising avenue for the clinical treatment of spinal cord injury.

  17. visual bone marrow mesenchymal stem cell transplantation in the repair of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Rui-ping Zhang; Cheng Xu; Yin Liu; Jian-ding Li; Jun Xie

    2015-01-01

    An important factor in improving functional recovery from spinal cord injury using stem cells is maximizing the number of transplanted cells at the lesion site. Here, we established a contusion model of spinal cord injury by dropping a weight onto the spinal cord at T7–8. Superparamagnet-ic iron oxide-labeled bone marrow mesenchymal stem cells were transplanted into the injured spinal cordvia the subarachnoid space. An outer magnetic ifeld was used to successfully guide the labeled cells to the lesion site. Prussian blue staining showed that more bone marrow mesen-chymal stem cells reached the lesion site in these rats than in those without magnetic guidance or superparamagnetic iron oxide labeling, and immunolfuorescence revealed a greater number of complete axons at the lesion site. Moreover, the Basso, Beattie and Bresnahan (BBB) locomotor rating scale scores were the highest in rats with superparamagnetic labeling and magnetic guid-ance. Our data conifrm that superparamagnetic iron oxide nanoparticles effectively label bone marrow mesenchymal stem cells and impart sufficient magnetism to respond to the external magnetic ifeld guides. More importantly, superparamagnetic iron oxide-labeled bone marrow mesenchymal stem cells can be dynamically and non-invasively trackedin vivo using magnetic resonance imaging. Superparamagnetic iron oxide labeling of bone marrow mesenchymal stem cells coupled with magnetic guidance offers a promising avenue for the clinical treatment of spinal cord injury.

  18. PLGA-encapsulated perfluorocarbon nanoparticles for simultaneous visualization of distinct cell populations by (19)F MRI

    NARCIS (Netherlands)

    Srinivas, M.; Tel, J.; Schreibelt, G.; Bonetto, F.J.; Cruz, L.J.; Amiri, H.; Heerschap, A.; Figdor, C.G.; Vries, I.J.M. de

    2015-01-01

    AIM: In vivo imaging using (19)F MRI is advantageous, due to its ability to quantify cell numbers, but is limited for a lack of suitable labels. Here, we formulate two stable and clinically applicable labels for tracking two populations of primary human dendritic cells (DCs) simultaneously. MATERIAL

  19. Visualization of the specific interaction of sulfonylurea-incorporated polymer with insulinoma cell line MIN6.

    Science.gov (United States)

    Park, Keun-Hong; Akaike, Toshihiro

    2004-02-01

    A derivative of sulfonylurea (SU) that mimics glibenclamide in chemical structure was synthesized and incorporated into a water-soluble polymeric backbone as a biospecific polymer for stimulating insulin secretion. In this study, a backbone polymer fluorescence-labeled with rodamine-B isothiocyanate was found to be strongly adsorbed onto MIN6 cells, probably due to its specific interaction mediated by SU receptors on the cell membrane. The intensity of fluorescence on the cells was significantly increased by increasing the incubation time and polymer concentration. To verify the specific interaction between the SU (K(+) channel closer)-incorporated copolymer and MIN6 cells, the cells were pretreated with diazoxide, an agonist of the ATP-sensitive K(+) channel (K(+) channel opener), before adding the polymer to the cell culture medium. This treatment suppressed the interaction between SU and MIN6 cells. A confocal laser microscopic study confirmed this effect. The results of this study provide evidence that SU-incorporated copolymer stimulates insulin secretion through the specific interactions of SU moieties in the polymer with MIN6 cells.

  20. Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse.

    Directory of Open Access Journals (Sweden)

    Marc Bajénoff

    Full Text Available Memory CD8(+ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m. Memory CD8(+ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+ T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+ T cells provide a local response by secreting effector molecules around infected cells.

  1. A water framework directive (WFD) compliant determination of eologically acceptable flows in alpine rivers - a river type specific approach

    Science.gov (United States)

    Jäger, Paul; Zitek, Andreas

    2010-05-01

    Currently the EU-Water Framework Directive (WFD) represents the driving force behind the assessment for rehabilitation and conservation of aquatic resources throughout Europe. Hydropower production, often considered as "green energy", in the past has put significant pressures on river systems like fragmentation by weirs, impoundment, hydropeaking and water abstraction. Due to the limited availability of data for determining ecologically acceptable flow for rivers at water abstraction sites, a special monitoring program was conducted in the federal state of Salzburg in Austria from 2006 to 2009. Water abstraction sites at 19 hydropower plants, mostly within the trout region of the River Salzach catchment, were assessed in detail with regard to the effect of water abstraction on fish and macrozoobenthos. Based on a detailed assessment of the specific local hydro-morphological and biological situations, the validity of natural low flow criteria (Absolute Minimum Flow - AMF, the lowest daily average flow ever measured and Mean Annual Daily Low Flow - MADLF) as starting points for the determination of an ecologically acceptable flow was tested. It was assessed, if a good ecological status in accordance with the EU-WFD can be maintained at natural AMF. Additionally it was tested, if important habitat parameters describing connectivity, river type specific flow variability and river type specific habitats are maintained at this discharge. Habitat modelling was applied in some situations. Hydraulic results showed that at AMF the highest flow velocity classes were lost in most situations. When AMF was significantly undercut, flow velocities between 0,0 - 0,4 m/s became dominant, describing the loss of the river type specific flow character, leading to a loss of river type specific flow variability and habitats and increased sedimentation of fines. Furthermore limits for parameters describing connectivity for fish like maximum depth at the pessimum profile and minimum flow

  2. Interactions of multiwalled carbon nanotubes with algal cells: quantification of association, visualization of uptake, and measurement of alterations in the composition of cells.

    Science.gov (United States)

    Rhiem, Stefan; Riding, Matthew J; Baumgartner, Werner; Martin, Francis L; Semple, Kirk T; Jones, Kevin C; Schäffer, Andreas; Maes, Hanna M

    2015-01-01

    Carbon nanotubes (CNTs) are considered promising materials in nanotechnology. We quantified CNT accumulation by the alga Desmodesmus subspicatus. Cells were exposed to radiolabeled CNTs ((14)C-CNTs;1 mg/L) to determine uptake and association, as well as elimination and dissociation in clear media.Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was used to detect effects of CNTs on algae. CNT-cell interactions were visualized by electron microscopy and related to alterations in their cell composition. A concentration factor of 5000 L/kg dry weight was calculated. Most of the material agglomerated around the cells, but single tubes were detected in the cytoplasm. Computational analyses of the ATR-FTIR data showed that CNT treated algae differed from controls at all sampling times.CNT exposure changed the biochemical composition of cells. The fact that CNTs are bioavailable for algae and that they influence the cell composition is important with regard to environmental risk assessment of this nanomaterial.

  3. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  4. Patterning processes in aggregates of Hydra cells visualized with the monoclonal antibody, TS19.

    Science.gov (United States)

    Sato, M; Bode, H R; Sawada, Y

    1990-10-01

    The monoclonal antibody, TS19, (Heimfeld et al., 1985), labels the apical surface of ectodermal epithelial cells of tentacles and lower peduncles in Hydra. To investigate the patterning process in a tissue whose original pattern was completely destroyed, the TS19 staining pattern was examined in developing aggregates of Hydra cells. Two types of aggregates were prepared. G-aggregates were made from tissue of the gastric portion of animals and RG-aggregates from gastric tissue allowed to regenerate for 24 hr before making aggregates. G-aggregates were initially TS19-negative, and later dim and uniformly TS19-positive. Thereafter, TS19 staining broke up into brightly stained and unstained regions. The brightly staining regions developed into head or foot structures. The TS19 pattern in RG-aggregates developed differently. Since the initial aggregates contained cells of regenerating tips, they started with TS19-positive cells as well as TS19-negative cells. The numbers of brightly staining TS19-positive cells increased with time. Some patches of these cells developed into head or foot structures, while others did not. These results and a simulation using a reaction-diffusion model suggest that the changes in activation levels affected the temporal changes in the pattern of TS19 staining, and that the de novo pattern formation in hydra can be explained in terms of a process involving activation and inhibition properties.

  5. Comparison of Narrowband Imaging with Autofluorescence Imaging for Endoscopic Visualization of Superficial Squamous Cell Carcinoma Lesions of the Esophagus

    Directory of Open Access Journals (Sweden)

    Haruhisa Suzuki

    2012-01-01

    Full Text Available Aim. To compare narrowband imaging (NBI and autofluorescence imaging (AFI endoscopic visualization for identifying superficial esophageal squamous cell carcinoma (SCC. Methods. Twenty-four patients with superficial esophageal carcinomas diagnosed at previous hospitals were enrolled in this study. Lesions were initially detected using white-light endoscopy and then observed with both NBI and AFI. Endoscopic images documented each method, and three endoscopists experienced in esophageal imaging retrospectively reviewed respective images of histologically confirmed esophageal SCCs. Images were assessed for quality in identifying superficial SCCs and rated as excellent, fair, or poor by the three reviewers with interobserver agreement calculated using kappa (κ statistics. Results. Thirty-one lesions histologically confirmed as superficial esophageal SCCs were detected in 24 patients. NBI images of 27 lesions (87% were rated as excellent, three as fair, and one as poor compared to AFI images of 19 lesions (61% rated as excellent, 10 as fair and two as poor (P<0.05. Moderate interobserver agreement (κ=0.42, 95% CI 0.24–0.60 resulted in NBI while fair agreement (κ=0.35, 95% CI 0.18–0.51 was achieved using AFI. Conclusion. NBI may be more effective than AFI for visualization of esophageal SCC.

  6. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    Science.gov (United States)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  7. Visualization and identification of IL-7 producing cells in reporter mice.

    Directory of Open Access Journals (Sweden)

    Renata I Mazzucchelli

    Full Text Available Interleukin-7 (IL-7 is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.

  8. Muscle fiber type specific activation of the slow myosin heavy chain 2 promoter by a non-canonical E-box.

    Science.gov (United States)

    Weimer, Kristina; DiMario, Joseph X

    2016-01-22

    Different mechanisms control skeletal muscle fiber type gene expression at specific times in vertebrate development. Embryonic myogenesis leading to formation of primary muscle fibers in avian species is largely directed by myoblast cell commitment to the formation of diverse fiber types. In contrast, development of different secondary fiber types during fetal myogenesis is partly determined by neural influences. In both primary and secondary chicken muscle fibers, differential expression of the slow myosin heavy chain 2 (MyHC2) gene distinguishes fast from fast/slow muscle fibers. This study focused on the transcriptional regulation of the slow MyHC2 gene in primary myotubes formed from distinct fast/slow and fast myogenic cell lineages. Promoter deletion analyses identified a discrete 86 bp promoter segment that conferred fiber type, lineage-specific gene expression in fast/slow versus fast myoblast derived primary myotubes. Sequence analysis and promoter activity assays determined that this segment contains two functional cis-regulatory elements. One element is a non-canonical E-box, and electromobility shift assays demonstrated that both cis-elements interacted with the E-protein, E47. The results indicate that primary muscle fiber type specific expression of the slow MyHC2 gene is controlled by a novel mechanism involving a transcriptional complex that includes E47 at a non-canonical E-box.

  9. Magnetically levitated nano-robots: an application to visualization of nerve cells injuries.

    Science.gov (United States)

    Lou, Mingji; Jonckheere, Edmond

    2007-01-01

    This paper proposes a swarm of magnetically levitated nano-robots with high sensitivity nano-sensors as a mean to detect chemical sources, specifically the chemical signals released by injured nervous cells. In the aftermath of the process, further observation by these nano-robots would be used to monitor the healing process and assess the amount of regeneration, if any, or even the repair, of the injured nervous cells.

  10. Construction of an electrochemical cell to visualize samples in situ in stereomicroscope

    OpenAIRE

    Mônica Alessandra Silva Alencar; Assis Vicente Benedetti; Cecílio Sadao Fugivara; Younès Messaddeq

    2010-01-01

    The electrochemical study of glass like tungsten oxide derivatives requires the construction of special electrodes due to the fact that these glasses are not conductive. Electrodes modified with WO3 change their color when submitted to some potential perturbation. The color change of the electrochromic materials was observed in situ by coupling an electrochemical cell to a stereomicroscope. The constructed cell is versatile and may represent a great contribution to the electrochemical studies...

  11. Fiber type specific expression of TNF-alpha, IL-6 and IL-18 in human skeletal muscles

    DEFF Research Database (Denmark)

    Plomgaard, Peter; Penkowa, Milena; Pedersen, Bente K

    2005-01-01

    Skeletal muscle is now recognized as an endocrine organ with the capacity to produce signal peptides in response to muscle contractions. Here we demonstrate that resting healthy human muscles express cytokines in a fiber type specific manner. Human muscle biopsies from seven healthy young males...... were obtained from m. triceps, m. quadriceps vastus lateralis and m. soleus. Type I fibers contributed (mean +/- SE) 24.0 +/- 2.5% in triceps of total fibers, 51.3 +/- 2.4% in vastus and 84.9 +/- 22% in soleus. As expected, differences in the fiber type composition were accompanied by marked...... differences between the three muscles with regard to MHC I and MHC IIa mRNA expression. Immunohistochemistry demonstrated that tumor necrosis factor (TNF)-alpha and interleukin (IL)-18 were solely expressed by type II fibers, whereas the expression of IL-6 was more prominent in type I compared to type II...

  12. Requirements for future control room and visualization features in the Web-of-Cells framework defined in the ELECTRA project

    DEFF Research Database (Denmark)

    Tornelli, Carlo; Zuelli, Roberto; Marinelli, Mattia

    2017-01-01

    This paper outlines an overview of the general requirements for the control rooms of the future power systems (2030+). The roles and activities in the future control centres will evolve with respect to the switching, dispatching and restoration functions currently active. The control centre...... operators will supervise on the power system and intervene - when necessary - thanks to the maturation and wide scale deployment of flexible controls. For the identification of control room requirements, general trends in power system evolution are considered and mainly the outcomes of the ELECTRA IRP...... project, that proposes a new Web-of-Cell (WoC) power system control architecture. Dedicated visualization features are proposed, aimed to support the control room operators activities in a WoC oriented approach. Furthermore, the work takes into account the point of view of network operators about future...

  13. Cell-Targeted Optogenetics and Electrical Microstimulation Reveal the Primate Koniocellular Projection to Supra-granular Visual Cortex.

    Science.gov (United States)

    Klein, Carsten; Evrard, Henry C; Shapcott, Katharine A; Haverkamp, Silke; Logothetis, Nikos K; Schmid, Michael C

    2016-04-01

    Electrical microstimulation and more recently optogenetics are widely used to map large-scale brain circuits. However, the neuronal specificity achieved with both methods is not well understood. Here we compare cell-targeted optogenetics and electrical microstimulation in the macaque monkey brain to functionally map the koniocellular lateral geniculate nucleus (LGN) projection to primary visual cortex (V1). Selective activation of the LGN konio neurons with CamK-specific optogenetics caused selective electrical current inflow in the supra-granular layers of V1. Electrical microstimulation targeted at LGN konio layers revealed the same supra-granular V1 activation pattern as the one elicited by optogenetics. Taken together, these findings establish a selective koniocellular LGN influence on V1 supra-granular layers, and they indicate comparable capacities of both stimulation methods to isolate thalamo-cortical circuits in the primate brain.

  14. Type-specific diagnosis and evaluation of longitudinal tumor extent of Borrmann type IV gastric cancer: CT versus gastroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Im [Dept. of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, Institute of Radiation Medicine, Seoul National University Medical Research Center, Seoul (Korea, Republic of); Kim, Young Hoon; Lee, Kyung Ho; Kim, So Yeon; Lee, Yoon Jin; Park, Young Soo; Kim, Na Young; Lee, Dong Ho; Kim, Hyung; Ho; Park, Do Joong; Lee, Hye Seung [Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam (Korea, Republic of)

    2013-08-15

    To compare the accuracy of computed tomography (CT) with that of gastroscopy for the extent of evaluation of longitudinal tumor and type-specific diagnosis of Borrmann type IV gastric cancer. Fifty-nine patients (35 men with mean age of 60 years and 24 women with mean age of 55 years) who underwent surgical resection of Borrmann type IV gastric cancer were included in this study. Histopathological analysis data was used as a reference standard to confirm the clinical interpretations of gastroscopy and CT for the diagnosis of Borrmann type IV and evaluation of longitudinal tumor extent. For the evaluation of longitudinal extent, gastroscopic and CT results were classified as underestimated, accurate, or overestimated. The McNemar test was used to identify statistically significant differences in the accuracy between gastroscopy and CT. For the diagnosis of Borrmann type IV gastric cancer, the accuracy of CT was significantly higher than that of gastroscopy (74.6% [44/59] vs. 44.1% [26/59], p < 0.001). CT was significantly more accurate in assessing the overall tumor extent than gastroscopy (61.4% [35/57] vs. 28.1% [16/57], p < 0.001). The proximal (75.4% [43/57] vs. 50.9% [29/57], p = 0.003) and distal tumor extent (71.9% [41/57] vs. 43.9% [25/57], p < 0.05) were more accurately predicted by CT compared with gastroscopy. The underestimation of tumor extent was a major source of error in both examinations. CT was found to be more predictive than gastroscopy in type-specific diagnosis and the evaluation of longitudinal tumor extent in patients with Borrmann type IV gastric cancer.

  15. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    Science.gov (United States)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  16. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

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

    Science.gov (United States)

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

    2016-02-01

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

  18. Giant lens, a gene involved in cell determination and axon guidance in the visual system of Drosophila melanogaster.

    Science.gov (United States)

    Kretzschmar, D; Brunner, A; Wiersdorff, V; Pflugfelder, G O; Heisenberg, M; Schneuwly, S

    1992-01-01

    Mutations in the Drosophila gene giant lens (gil) affect ommatidial development, photoreceptor axon guidance and optic lobe development. We have cloned the gene using an enhancer trap line. Molecular analysis of gil suggests that it encodes a secreted protein with an epidermal-growth-factor-like motif. We have generated mutations at the gil locus by imprecise excision of the enhancer trap P-element. In the absence of gil, additional photoreceptors develop at the expense of pigment cells, suggesting an involvement of gil in cell determination during eye development. In addition, gil mutants show drastic effects on photoreceptor axon guidance and optic lobe development. In wildtype flies, photoreceptor axons grow from the eye disc through the optic stalk into the larval brain hemisphere, where retinal innervation is required for the normal development of the lamina and distal medulla. The projection pattern of these axons in the developing lamina and medulla is highly regular and reproducible. In gil, photoreceptor axons enter the larval brain but fail to establish proper connections in the lamina or medulla. We propose that gil encodes a new type of signalling molecule involved in the process of axon pathfinding and cell determination in the visual system of Drosophila. Images PMID:1628618

  19. Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging.

    Science.gov (United States)

    Annibale, Paolo; Gratton, Enrico

    2015-03-19

    Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.

  20. Visualization of Hierarchical Nanodomains in Polymer/Fullerene Bulk Heterojunction Solar Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Jianguo; Miller, Dean J.; Chen, Wei; Xu, Tao; Yu, L; Darling, Seth B.; Zaluzec, Nestor J.

    2014-10-01

    raditional electron microscopy techniques such as bright-field imaging provide poor contrast for organic films and identification of structures in amorphous material can be problematic, particularly in high- performance organic solar cells. By combining energy-filtered corrected transmission electron microscopy, together with electron energy loss and X-ray energy-dispersive hyperspectral imaging, we have imaged PTB7/ PC61BM blended polymer optical photovoltaic films, and were able to identify domains ranging in size from several hundred nanometers to several nanometers in extent. This work verifies that microstructural domains exist in bulk heterojunctions in PTB7/PC61BM polymeric solar cells at multiple length scales and expands our understanding of optimal device performance providing insight for the design of even higher performance cells.

  1. Real-time visualization of prion transport in single live cells using quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Kan [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Li, Shu [AIDS Research Centre, Institute of Pathogen Biology, Chinese Academy of Medical Science, Beijing 100730 (China); Xie, Min [College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Wu, Di; Wang, WenXi; Chen, Rui; Huang, Liqin; Huang, Tao [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Pang, Daiwen, E-mail: dwpang@whu.edu.cn [College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

    2010-04-09

    Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP{sup C} to the infectious scrapie isoform PrP{sup Sc}. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP{sup C} to the cell membrane and in initiating PrP{sup C} endocytosis.

  2. VISUALIZATION OF DYNAMIC ORGANIZATION OF CYTOSKELETON GELS IN LIVING CELLS BY HYBRID—SPM

    Institute of Scientific and Technical Information of China (English)

    K.Kawabata; Y.Sado; M.Nagayama; T.Nitta; K.Nemoto; Y.Koyama; H.Haga

    2003-01-01

    We succeeded in performing of hybrid Scanning Probe Microscopy(hybrid-SPM) in which mechanical-SPM and fluorescence microscopy are combined.This technique is able to measure simultaneously mechanical properties and distribution of cytoskeletons of lining cells by using green fluorescent protein.We measured evolution of both local elasticity and distributions of actin stress fibers in an identical fibroblast living in physiological conditions.The SPM experiments revealed that stiffer lines develop in living cells,which correspond to actin stress fibers.The elasticity of the actin stress fibers is as high as 100kPa.We discuss mechanical effects on the development of actin filament networks.

  3. VISUALIZATION OF DYNAMIC ORGANIZATION OF CYTOSKELETON GELS IN LIVING CELLS BY HYBRID-SPM

    Institute of Scientific and Technical Information of China (English)

    K.Kawabata; Y.Sado; M.Nagayama; T.Nitta; K.Nemoto; Y.Koyama; H.Haga

    2003-01-01

    We succeeded in performing of hybrid Scanning Probe Microscopy (hybrid-SPM) in which mechanical-SPM and fluorescence microscopy are combined. This technique is able to measure simultaneously mechanical properties and distribution of cytoskeletons of living cells by using green fluorescent protein. We measured evolution of both local elasticity and distributions of actin stress fibers in an identical fibroblast living in physiological conditions. The SPM experiments revealed that stiffer lines develop in living cells, which correspond to actin stress fibers. The elasticity of the actin stress fibers is as high as 100 kPa. We discuss mechanical effects on the development of actin filament networks.

  4. Visualizing the molecular sociology at the HeLa cell nuclear periphery

    NARCIS (Netherlands)

    Mahamid, Julia; Pfeffer, Stefan; Schaffer, Miroslava; Villa, Elizabeth; Danev, Radostin; Cuellar, Luis Kuhn; Förster, Friedrich; Hyman, Anthony A; Plitzko, Jürgen M; Baumeister, Wolfgang

    2016-01-01

    The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed th

  5. Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

    Directory of Open Access Journals (Sweden)

    Alexandre S. Boutorine

    2013-12-01

    Full Text Available This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i sequence-specific peptides and proteins; (ii triplex-forming oligonucleotides and (iii polyamide oligo(N-methylpyrrole/N-methylimidazole minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

  6. MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain

    DEFF Research Database (Denmark)

    Vreys, Ruth; Vande Velde, Greetje; Krylychkina, Olga

    2010-01-01

    neurogenesis. Quantitative analysis of bromodeoxyuridine labeled cells revealed altered proliferation in the SVZ and NPC migration after in situ MPIO injection. From the labeling strategies presented in this report, intraventricular injection of a small number of MPIOs combined with the transfection agent poly...... the impact on adult neurogenesis when new in situ labeling strategies are developed....

  7. Visualizing pancreatic {beta}-cell mass with [{sup 11}C]DTBZ

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Norman Ray [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Souza, Fabiola [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Witkowski, Piotr [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Maffei, Antonella [Institute of Genetics and Biophysics ' Adriano Buzzati-Traverso' , CNR, Naples 80131 (Italy); Raffo, Anthony [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Herron, Alan [Center for Comparative Medicine and The Department of Pathology, Baylor College of Medicine, Houston, TX 77030 (United States); Kilbourn, Michael [Department of Radiology, University of Michigan, Ann Arbor, MI 48109-0638 (United States); Jurewicz, Agata [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Herold, Kevan [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States); Liu, Eric [Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, MD 20854 (United States); Hardy, Mark Adam [Department of Medicine, Columbia University Medical School, New York, NY 10032 (United States); Van Heertum, Ronald [Department of Radiology, Columbia University Medical School, New York, NY 10032 (United States); Harris, Paul Emerson [Department of Surgery, Columbia University Medical School, New York, NY 10032 (United States)]. E-mail: peh1@columbia.edu

    2006-10-15

    {beta}-Cell mass (BCM) influences the total amount of insulin secreted, varies by individual and by the degree of insulin resistance, and is affected by physiologic and pathologic conditions. The islets of Langerhans, however, appear to have a reserve capacity of insulin secretion and, overall, assessments of insulin and blood glucose levels remain poor measures of BCM, {beta}-cell function and progression of diabetes. Thus, novel noninvasive determinations of BCM are needed to provide a quantitative endpoint for novel therapies of diabetes, islet regeneration and transplantation. Built on previous gene expression studies, we tested the hypothesis that the targeting of vesicular monoamine transporter 2 (VMAT2), which is expressed by {beta} cells, with [{sup 11}C]dihydrotetrabenazine ([{sup 11}C]DTBZ), a radioligand specific for VMAT2, and the use of positron emission tomography (PET) can provide a measure of BCM. In this report, we demonstrate decreased radioligand uptake within the pancreas of Lewis rats with streptozotocin-induced diabetes relative to their euglycemic historical controls. These studies suggest that quantitation of VMAT2 expression in {beta} cells with the use of [{sup 11}C]DTBZ and PET represents a method for noninvasive longitudinal estimates of changes in BCM that may be useful in the study and treatment of diabetes.

  8. Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR

    DEFF Research Database (Denmark)

    Tolker-Nielsen, Tim; Holmstrøm, Kim; Molin, Søren

    1997-01-01

    reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect...

  9. Three-dimensional visualization of virus-infected cells by serial sectioning: an electron microscopic study using resin embedded cells.

    Science.gov (United States)

    Schauflinger, Martin; Villinger, Clarissa; Walther, Paul

    2013-01-01

    In this paper we show how to obtain a three-dimensional model of virus-infected cells by serial sectioning of resin embedded samples and transmission electron microscopic imaging. The method bases on sample fixation by high pressure freezing and processing by freeze substitution with the goal to preserve the structures of interest close to the natural state, as previously described (Walther et al., High pressure freezing for scanning transmission electron tomography analysis of cellular organelles. In: Mossman BT, Taatjes DJ (eds) Cell imaging techniques, vol 931, Methods in molecular biology. Humana Press, Totowa, NJ, pp 525-535, 2013). Advantages of serial sectioning compared to that of other tomographic methods are as follows: No special and expensive additional equipment is required. Relatively large volumes, such as whole cells, can be three-dimensionally reconstructed in a reasonable amount of time. Serial sectioning is a non-destructive method; the sections can be stored, re-imaged, or processed for immunogold labeling when more specific data are requested or when new scientific questions are raised (e.g., higher magnifications, protein distributions). We have recently used this method to obtain a three-dimensional model of the complete assembly complex of an HCMV infected cell, which allowed a detailed insight into this virally induced compartment (Schauflinger et al., Cell Microbiol 15(2):305-314, 2013).

  10. Simultaneous visualization of oxygen partial pressure, current density, and water droplets in serpentine fuel cell during power generation for understanding reaction distributions

    Science.gov (United States)

    Takanohashi, Kazuhiro; Suga, Takeo; Uchida, Makoto; Ueda, Toshihide; Nagumo, Yuzo; Inukai, Junji; Nishide, Hiroyuki; Watanabe, Masahiro

    2017-03-01

    Understanding the reaction distributions inside a polymer electrolyte fuel cell (PEFC) is essential for the higher performance and durability. We have developed a new see-through cell and visualized the distributions of oxygen partial pressure and current density inside a running PEFC at the temperature of 40 and 80 °C and the relative humidity of 53%. The oxygen utilization was changed from 0% to 80% by changing the current density. At higher oxygen utilizations, the current density was higher and therefore the water generation. Generated water droplets in the flow channel were also visualized, allowing for the simultaneous visualization of the distribution of the oxygen partial pressure, current density, and water droplets. By combining the observations of all three parameters, the reactions inside a membrane-electrode assembly were discussed.

  11. Visualizing Single Cell Biology: Nanosims Studies of Carbon and Nitrogen Metabolism in Diazotrophic Cyanobacteria

    Science.gov (United States)

    Pett-Ridge, J.; Finzi, J. A.; Capone, D. G.; Popa, R.; Nealson, K. H.; Ng, W.; Spormann, A. M.; Hutcheon, I. D.; Weber, P. K.

    2007-12-01

    Filamentous nitrogen fixing (diazotrophic) cyanobacteria are key players in global nutrient cycling, but the relationship between CO2- and N2-fixation and intercellular exchange of these elements remains poorly understood in many genera. These bacteria are faced with the challenge of isolating regions of N-fixation (O2 inhibited) and photosynthetic (O2 producing) activity. We used isotope labeling in conjunction with a high-resolution isotope and elemental mapping technique (NanoSIMS) to quantitatively describe 13C and 15N uptake and transport in two aquatic cyanobacteria grown on NaH13CO3 and 15N2. The technical challenges of tracing isotopes within individual bacteria can be overcome with high resolution Secondary Ion Mass Spectrometry (NanoSIMS). In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio 'map' can then be generated for the analyzed area. Using sequentially harvested cyanobacteria in conjunction with enriched H13CO3 and 15N2 incubations, we measured temporal enrichment patterns that evolve over the course of a day's growth and suggest tightly regulated changes in fixation kinetics. With a combination of TEM, SEM and NanoSIMS analyses, we also mapped the distribution of C, N and Mo (a critical nitrogenase co-factor) isotopes in intact cells. Our results suggest that NanoSIMS mapping of metal enzyme co-factors may be a powerful method of identifying physiological and morphological characteristics within individual bacterial cells, and could be used to provide a 3-dimensional context for more traditional analyses such as immunogold labeling. Finally, we resolved patterns of isotope enrichment at multiple spatial scales: sub-cellular variation, cell-cell differences along filaments

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

    Science.gov (United States)

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

    2014-06-01

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

  13. High-definition optical coherence tomography enables visualization of individual cells in healthy skin

    DEFF Research Database (Denmark)

    Boone, Marc; Jemec, Gregor B E; Del Marmol, Véronique

    2012-01-01

    of the HD-OCT could be confirmed by the phantom analysis. The identification of cells in the epidermis can be made by both techniques. RCM offers the best lateral resolution, and HD-OCT has the best penetration depth, providing images of individual cells deeper within the dermis. Eccrine ducts and hair......High-definition OCT (HD-OCT) is an innovative technique based on the principle of conventional OCT. Our objective was to test the resolution and image quality of HD-OCT in comparison with reflectance confocal microscopy (RCM) of healthy skin. Firstly, images have been made of a ultra......-high-resolution line-pair phantome with both systems. Secondly, we investigated 21 healthy volunteers of different phototypes with HD-OCT and RCM on volar forearm and compared the generated images. HD-OCT displays also differences depending on the skin phototype and anatomical site. The 3-μm lateral resolution...

  14. Visual Servoing for Optimization of Anticancer Drug Uptake in Human Breast Cancer Cells

    Science.gov (United States)

    2000-09-01

    tumor cells. Cancer Research. 57:1590-1596. DeVita , V. T., Jr. 1997. Cancer: principles and practice of oncology . Lippincott-Raven, Philadelphia. Ethier...vitro assays to select chemotherapy regimens tailored to the tumor of the individual patient is not new ( DeVita , 1997). A summary of clinical correlations...pooled from several individual studies ( DeVita , 1997). These authors have suggested that these assays be referred to as drug-response assays, rather than

  15. Visualization of Fuel Cell Water Transport and Performance Characterization under Freezing Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Kandlikar, Satish G. [Rochester Inst. of Technology, Rochester, NY (United States); Lu, Zijie [Rochester Inst. of Technology, Rochester, NY (United States); Rao, Navalgund [Rochester Inst. of Technology, Rochester, NY (United States); Sergi, Jacqueline [Rochester Inst. of Technology, Rochester, NY (United States); Rath, Cody [Rochester Inst. of Technology, Rochester, NY (United States); McDade, Christopher [Rochester Inst. of Technology, Rochester, NY (United States); Trabold, Thomas [General Motors, Honeoye Falls, NY (United States); Owejan, Jon [General Motors, Honeoye Falls, NY (United States); Gagliardo, Jeffrey [General Motors, Honeoye Falls, NY (United States); Allen, Jeffrey [Michigan Technological Univ., Houghton, MI (United States); Yassar, Reza S. [Michigan Technological Univ., Houghton, MI (United States); Medici, Ezequiel [Michigan Technological Univ., Houghton, MI (United States); Herescu, Alexandru [Michigan Technological Univ., Houghton, MI (United States)

    2010-05-30

    In this program, Rochester Institute of Technology (RIT), General Motors (GM) and Michigan Technological University (MTU) have focused on fundamental studies that address water transport, accumulation and mitigation processes in the gas diffusion layer and flow field channels of the bipolar plate. These studies have been conducted with a particular emphasis on understanding the key transport phenomena which control fuel cell operation under freezing conditions.

  16. Current techniques for visualizing RNA in cells [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Lilith V.J.C. Mannack

    2016-04-01

    Full Text Available Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations.

  17. Visual agnosia.

    Science.gov (United States)

    Álvarez, R; Masjuan, J

    2016-03-01

    Visual agnosia is defined as an impairment of object recognition, in the absence of visual acuity or cognitive dysfunction that would explain this impairment. This condition is caused by lesions in the visual association cortex, sparing primary visual cortex. There are 2 main pathways that process visual information: the ventral stream, tasked with object recognition, and the dorsal stream, in charge of locating objects in space. Visual agnosia can therefore be divided into 2 major groups depending on which of the two streams is damaged. The aim of this article is to conduct a narrative review of the various visual agnosia syndromes, including recent developments in a number of these syndromes.

  18. Direct visualization of neo-vessel formation following peripheral injection of bone marrow derived CD34+ cells in experimental myocardial damage.

    Science.gov (United States)

    Ciulla, M M; Ferrero, S; Gianelli, U; Paliotti, R; Magrini, F; Braidotti, P

    2007-01-01

    The definitive fate of peripherally injected PKH26 labelled bone marrow mononuclear cells expressing the CD34+ antigen following experimental myocardial cryodamage in rats (n=10) has been examined by direct visualization on photoconverted light and electron microscopy images. One week after the injection in each rat of about 150,000 CD34+ cells early stage PKH26+ vascular structures were localized in the infarcted areas, suggesting that a potential benefit of this therapeutic approach consists in the regeneration of the vasculature.

  19. NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System.

    Directory of Open Access Journals (Sweden)

    Louis C Leung

    Full Text Available Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC, a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway.

  20. Dynamic Visualization of mTORC1 Activity in Living Cells

    Science.gov (United States)

    Zhou, Xin; Clister, Terri L.; Lowry, Pamela R.; Seldin, Marcus M.; Wong, G. William; Zhang, Jin

    2015-01-01

    Summary The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. It has become increasingly clear that mTORC1 activity is regulated in time and space inside the cell, but direct interrogation of such spatiotemporal regulation is challenging. Here we describe a genetically encoded mTORC1 activity reporter (TORCAR) that exhibits a change in FRET in response to phosphorylation by mTORC1. Co-imaging mTORC1 activity and calcium dynamics revealed that a growth factor-induced calcium transient contributes to mTORC1 activity. Dynamic activity maps generated using subcellularly targeted TORCAR uncovered mTORC1 activity not only in cytosol and at the lysosome but also in nucleus and at the plasma membrane. Furthermore, a wide distribution of activities was observed upon growth factor stimulation, whereas leucine ester, an amino acid surrogate, induces more compartmentalized activities at the lysosome and in nucleus. Thus, mTORC1 activities are spatiotemporally regulated in a signal-specific manner. PMID:25772363

  1. Dynamic Visualization of mTORC1 Activity in Living Cells

    Directory of Open Access Journals (Sweden)

    Xin Zhou

    2015-03-01

    Full Text Available The mechanistic target of rapamycin complex 1 (mTORC1 senses diverse signals to regulate cell growth and metabolism. It has become increasingly clear that mTORC1 activity is regulated in time and space inside the cell, but direct interrogation of such spatiotemporal regulation is challenging. Here, we describe a genetically encoded mTORC1 activity reporter (TORCAR that exhibits a change in FRET in response to phosphorylation by mTORC1. Co-imaging mTORC1 activity and calcium dynamics revealed that a growth-factor-induced calcium transient contributes to mTORC1 activity. Dynamic activity maps generated with the use of subcellularly targeted TORCAR uncovered mTORC1 activity not only in cytosol and at the lysosome but also in the nucleus and at the plasma membrane. Furthermore, a wide distribution of activities was observed upon growth factor stimulation, whereas leucine ester, an amino acid surrogate, induces more compartmentalized activities at the lysosome and in the nucleus. Thus, mTORC1 activities are spatiotemporally regulated in a signal-specific manner.

  2. Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

    Directory of Open Access Journals (Sweden)

    Robert M. Martin

    2013-09-01

    Full Text Available Removal of introns from pre-messenger RNAs (pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py tract in mouse immunoglobulin μ (IgM pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context.

  3. Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci.

    Science.gov (United States)

    Zheng, Po-Xing; Chan, Yuen-Chi; Chiou, Chien-Shun; Chiang-Ni, Chuan; Wang, Shu-Ying; Tsai, Pei-Jane; Chuang, Woei-Jer; Lin, Yee-Shin; Liu, Ching-Chuan; Wu, Jiunn-Jong

    2015-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas) and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson's index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.

  4. Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci.

    Directory of Open Access Journals (Sweden)

    Po-Xing Zheng

    Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson's index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.

  5. Species-specific and mating type-specific DNA regions adjacent to mating type idiomorphs in the genus Neurospora.

    Science.gov (United States)

    Randall, T A; Metzenberg, R L

    1995-09-01

    Mating type idiomorphs control mating and subsequent sexual development in Neurospora crassa and were previously shown to be well conserved in other Neurospora species. The centromere-proximal flanks of the A and a idiomorphs, but not the distal flanks from representative heterothallic, pseudohomothallic, and homothallic Neurospora species contain apparent species-specific and/or mating type-specific sequences adjacent to the well-conserved idiomorphs. The variable flank is bordered by regions that are highly homologous in all species. The sequence of approximately 1 kb immediately flanking the conserved idiomorphs of each species was determined. Sequence identity between species ranged from 20% (essentially unrelated) to > 90%. By contrast, the mt-A1 gene shows 88-98% identity. Sequence and hybridization data also show that the centromere-proximal flanks are very different between the two mating types for N. intermedia, N. discreta, and N. tetrasperma, but not for N. sitophila and N. crassa. The data suggest a close evolutionary relationship between several of the species; this is suppported by phylogenetic analysis of their respective mt-A1 genes. The origin of the variable regions adjacent to the evolutionarily conserved mating type idiomorphs is unknown.

  6. Dynamics of bacterial communities in two unpolluted soils after spiking with phenanthrene: soil type specific and common responders

    Directory of Open Access Journals (Sweden)

    Guo-Chun eDing

    2012-08-01

    Full Text Available Considering their key role for ecosystem processes, it is important to understand the response of microbial communities in unpolluted soils to pollution with polycyclic aromatic hydrocarbons (PAH. Phenanthrene, a model compound for PAH, was spiked to a Cambisol and a Luvisol soil. Total community DNA from phenanthrene-spiked and control soils collected on days 0, 21 and 63 were analyzed based on PCR-amplified 16S rRNA genefragments. Denaturing gradient gel electrophoresis (DGGE fingerprints of bacterial communities increasingly deviated with time between spiked and control soils. In taxon specific DGGE, significant responses of Alphaproteobacteria and Actinobacteria became only detectable after 63 days, while significant effects on Betaproteobacteria were detectable in both soils after 21 days. Comparison of the taxonomic distribution of bacteria in spiked and control soils on day 63 as revealed by pyrosequencing indicated soil type specific negative effects of phenanthrene on several taxa, many of them belonging to the Gamma-, Beta- or Deltaproteobacteria. Bacterial richness and evenness decreased in spiked soils. Despite the significant differences in the bacterial community structure between both soils on day 0, similar genera increased in relative abundance after PAH spiking, especially Sphingomonas and Polaromonas. However, this did not result in an increased overall similarity of the bacterial communities in both soils.

  7. Comprehensive analysis of ultrasonic vocalizations in a mouse model of fragile X syndrome reveals limited, call type specific deficits.

    Directory of Open Access Journals (Sweden)

    Snigdha Roy

    Full Text Available Fragile X syndrome (FXS is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1 gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP. Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.

  8. Visual art and visual perception

    NARCIS (Netherlands)

    Koenderink, Jan J.

    2015-01-01

    Visual art and visual perception ‘Visual art’ has become a minor cul-de-sac orthogonal to THE ART of the museum directors and billionaire collectors. THE ART is conceptual, instead of visual. Among its cherished items are the tins of artist’s shit (Piero Manzoni, 1961, Merda d’Artista) “worth their

  9. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    Science.gov (United States)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active

  10. Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major.

    Directory of Open Access Journals (Sweden)

    Eric Muraille

    Full Text Available Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and

  11. Flow visualization

    CERN Document Server

    Merzkirch, Wolfgang

    1974-01-01

    Flow Visualization describes the most widely used methods for visualizing flows. Flow visualization evaluates certain properties of a flow field directly accessible to visual perception. Organized into five chapters, this book first presents the methods that create a visible flow pattern that could be investigated by visual inspection, such as simple dye and density-sensitive visualization methods. It then deals with the application of electron beams and streaming birefringence. Optical methods for compressible flows, hydraulic analogy, and high-speed photography are discussed in other cha

  12. Behavior of parasite-specific effector CD8+ T cells in the brain and visualization of a kinesis-associated system of reticular fibers.

    Science.gov (United States)

    Wilson, Emma H; Harris, Tajie H; Mrass, Paulus; John, Beena; Tait, Elia D; Wu, Gregory F; Pepper, Marion; Wherry, E John; Dzierzinski, Florence; Roos, David; Haydon, Philip G; Laufer, Terri M; Weninger, Wolfgang; Hunter, Christopher A

    2009-02-20

    To understand lymphocyte behavior in the brain, we used two-photon microscopy to visualize effector CD8(+) T cells during toxoplasmic encephalitis. These cells displayed multiple behaviors with two distinct populations of cells apparent: one with a constrained pattern of migration and one with a highly migratory subset. The proportion of these populations varied over time associated with changes in antigen availability as well as T cell expression of the inhibitory receptor PD1. Unexpectedly, the movement of infiltrating cells was closely associated with an infection-induced reticular system of fibers. This observation suggests that, whereas in other tissues pre-existing scaffolds exist that guide lymphocyte migration, in the brain specialized structures are induced by inflammation that guide migration of T cells in this immune-privileged environment.

  13. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    Science.gov (United States)

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  14. Visual Servoing

    OpenAIRE

    Chaumette, Francois; Hutchinson, Seth; Corke, Peter

    2016-01-01

    International audience; This chapter introduces visual servo control, using computer vision data in the servo loop to control the motion of a robot. We first describe the basic techniques that are by now well established in the field. We give a general overview of the formulation of the visual servo control problem, and describe the two archetypal visual servo control schemes: image-based and pose-based visual servo control. We then discuss performance and stability issues that pertain to the...

  15. Roles of Treg/Th17 Cell Imbalance and Neuronal Damage in the Visual Dysfunction Observed in Experimental Autoimmune Optic Neuritis Chronologically.

    Science.gov (United States)

    Liu, Yuanyuan; You, Caiyun; Zhang, Zhuhong; Zhang, Jingkai; Yan, Hua

    2015-12-01

    Optic neuritis associated with multiple sclerosis and its animal model, experimental autoimmune optic neuritis (EAON), is characterized by inflammation, T cell activation, demyelination, and neuronal damage, which might induce permanent vision loss. Elucidating the chronological relationship among the features is critical for treatment of demyelinating optic neuritis. EAON was induced in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein subcutaneously, and visual function was assessed by flash-visual evoked potential (F-VEP) at days 7, 11, 14, 19, 23, 28 post-immunization. Retinal ganglion cell (RGC) apoptosis was measured by terminal-deoxynucleotidyl transferase-mediated nick-end labeling. Demyelination and axonal damage were verified with myelin basic protein (MBP) and β-amyloid precursor protein staining, respectively. Real-time polymerase chain reaction quantified IL-17, IL-1β, TGF-β, FoxP3, IL-6, and IL-10 mRNA expression in the optic nerve, as well as FoxP3 and IL-17 staining. Systemic changes of Th17 and Treg cells were tested by flow cytometry in spleen. F-VEP latency was prolonged at 11 days and peaked at 23 days commensurate with demyelination. However, F-VEP amplitude was reduced at 11 days, preceding axon damage, and was exacerbated at 23 days when a peak in RGC apoptosis was detected. Th17 cells up-regulated as early as 7 days and peaked at 11 days, while Treg cells down-regulated inversely compared to Th17 cells change as verified by IL-17 and FoxP3 expression; spleen cell samples were slightly different, demonstrating marked changed at 14 days. Treg/Th17 cell imbalance in the optic nerve precedes and may initiate neuronal damage of axons and RGCs. These changes are commensurate with the appearances of visual dysfunction reflected in F-VEP and hence may offer a novel therapeutic avenue for vision preservation.

  16. Application of an enzyme-labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes, in recurrent tonsillitis.

    Science.gov (United States)

    Onouchi, Takanori; Mizutani, Yasuyoshi; Shiogama, Kazuya; Inada, Ken-ichi; Okada, Tatsuyoshi; Naito, Kensei; Tsutsumi, Yutaka

    2015-01-01

    Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme-labeled antigen method is a novel histochemical technique that visualizes specific antibody-producing cells in tissue sections by employing a biotin-labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde-fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme-labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR-detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti-Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A-reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.

  17. Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci).

    Science.gov (United States)

    Goto, Tatsuaki; Kaida, Atsushi; Miura, Masahiko

    2015-12-10

    Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation.

  18. Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

    Science.gov (United States)

    Yokawa, Satoru; Suzuki, Takahiro; Inouye, Satoshi; Inoh, Yoshikazu; Suzuki, Ryo; Kanamori, Takao; Furuno, Tadahide; Hirashima, Naohide

    2017-04-15

    We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

  19. Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells.

    Science.gov (United States)

    Staehelin, L A; Chapman, R L

    1987-05-01

    Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed

  20. Retinal lesions induce fast intrinsic cortical plasticity in adult mouse visual system.

    Science.gov (United States)

    Smolders, Katrien; Vreysen, Samme; Laramée, Marie-Eve; Cuyvers, Annemie; Hu, Tjing-Tjing; Van Brussel, Leen; Eysel, Ulf T; Nys, Julie; Arckens, Lutgarde

    2016-09-01

    Neuronal activity plays an important role in the development and structural-functional maintenance of the brain as well as in its life-long plastic response to changes in sensory stimulation. We characterized the impact of unilateral 15° laser lesions in the temporal lower visual field of the retina, on visually driven neuronal activity in the afferent visual pathway of adult mice using in situ hybridization for the activity reporter gene zif268. In the first days post-lesion, we detected a discrete zone of reduced zif268 expression in the contralateral hemisphere, spanning the border between the monocular segment of the primary visual cortex (V1) with extrastriate visual area V2M. We could not detect a clear lesion projection zone (LPZ) in areas lateral to V1 whereas medial to V2M, agranular and granular retrosplenial cortex showed decreased zif268 levels over their full extent. All affected areas displayed a return to normal zif268 levels, and this was faster in higher order visual areas than in V1. The lesion did, however, induce a permanent LPZ in the retinorecipient layers of the superior colliculus. We identified a retinotopy-based intrinsic capacity of adult mouse visual cortex to recover from restricted vision loss, with recovery speed reflecting the areal cortical magnification factor. Our observations predict incomplete visual field representations for areas lateral to V1 vs. lack of retinotopic organization for areas medial to V2M. The validation of this mouse model paves the way for future interrogations of cortical region- and cell-type-specific contributions to functional recovery, up to microcircuit level.

  1. Data visualization

    CERN Document Server

    Azzam, Tarek

    2013-01-01

    Do you communicate data and information to stakeholders? In Part 1, we introduce recent developments in the quantitative and qualitative data visualization field and provide a historical perspective on data visualization, its potential role in evaluation practice, and future directions. Part 2 delivers concrete suggestions for optimally using data visualization in evaluation, as well as suggestions for best practices in data visualization design. It focuses on specific quantitative and qualitative data visualization approaches that include data dashboards, graphic recording, and geographic information systems (GIS). Readers will get a step-by-step process for designing an effective data dashboard system for programs and organizations, and various suggestions to improve their utility.

  2. A Comparison of Pain Assessment Measures in Pediatric Sickle Cell Disease: Visual Analog Scale Versus Numeric Rating Scale.

    Science.gov (United States)

    Myrvik, Matthew P; Drendel, Amy L; Brandow, Amanda M; Yan, Ke; Hoffmann, Raymond G; Panepinto, Julie A

    2015-04-01

    Given the availability of various pain severity scales, greater understanding of the agreement between pain scales is warranted. We compared Visual Analog Scale (VAS) and Numeric Rating Scale (NRS) pain severity ratings in children with sickle cell disease (SCD) to identify the relationship and agreement between pain scale ratings. Twenty-eight patients (mean ± SD age, 14.65 ± 3.12 y, 50% female) receiving pain interventions within the emergency department completed serial VAS and NRS pain severity ratings every 30 minutes. Data were used to calculate the relationship (Spearman correlation) and agreement (Bland-Altman approach) between the VAS and NRS. One hundred twenty-eight paired VAS-NRS measurements were obtained. VAS and NRS ratings were significantly correlated for the initial assessment (rs = 0.88, P < 0.001) and all assessments (rs = 0.87, P < 0.001). Differences between VAS and NRS means were -0.52 (P = 0.006) for the initial assessment and -0.86 (P < 0.001) across all assessments. The difference between VAS and NRS ratings decreased as pain severity increased across all assessments (P = 0.027), but not the initial assessment. Within pediatric patients with SCD, VAS and NRS ratings were found to trend together; however, VAS scores were found to be significantly lower than NRS scores across assessments. The agreement between the 2 measures improved at increasing levels of pain severity. These findings demonstrate that the VAS and NRS are similar, but cannot be used interchangeably when assessing self-reported pain in SCD.

  3. A neural network model on self-organizing emergence of simple-cell receptive field with orientation selectivity in visual cortex

    Institute of Scientific and Technical Information of China (English)

    YANG; Qian(

    2001-01-01

    [1]Hubel, D. H.. Wiesel. T. N., Receptive fields of single neuron in the cat striate cortex, Journal of Physiology, 1959, 148:574-591.[2]Hubel. D. H.. Wiesel, T. N., Functional architecture macaque monkey visual cortexm, Proc. Roy. Soc. B, 1977, 198: 1-59.[3]Shou. T. D., Brain Mechanisms of Visual Information Processing (in Chinese), Shanghai: Shanghai Science-Technology and Education Press, 1997, 188-197.[4]Ferster, D., Chung, S., Wheat, H., Orientation selectivity of thalamic input to simple cells of cat visual cortex, Nature,1996, 380: 249-252.[5]Vidyasagar. T. R., Pei, X., Volgushev, M., Multiple mechanisms underlying the orientation selectivity of visual cortical neurons. TINS, 1996, 19: 272-277.[6]Artun, O. B., Shouval, H. Z., Cooper, L. N., The effect of dynamic synapses on spatiotemporal receptive fields in visual cortex, Proc. Natl. Acad. Sci. USA, 1998, 95:11999-12003.[7]Rolls, E. T.. Tovee, M. J., Sparseness of the neuronal representation of stimuli in the primate temporal visual cortex, J.Neurophysiology, 1995,73: 713-726.[8]Olshausen. B. A.. Field, D. J., Sparse coding with an overcomplete basis set: A strategy employed by V 1 ? Vision Research,1997.37: 3311-3325.[9]Bell. A. J., Sejnoswski, T. J., The "Independent components" of natural scenes are edge filters, Vision Research, 1997, 37:3327-3338.[10]Dan, Y., Atick, J. J., Reid, R. C., Efficient coding of natural scenes in the lateral geniculate nucleus: experimental test of a computational theory, Journal of Neuroscience, 1996, 16:3351-3362.[11]Field. D. J., Relations between the statistics of natural images and the response properties of cortical cells, Journal of the Optical Society of America A, 1987, 4: 2379-2394.[12]DeAngelis, G. C., Ohzawa, I., Freeman, R. D., Receptive filed dynamics in the central visual pathway, TINS, 1995,18:451-458.[13]Wang, Y. J., Qi, X. L., Chen, Y. Z., Simulations of receptive fields dynamics, TINS, 1996, 19: 385-386.

  4. BINDING TO AND RETENTION BY MUCOSAL CELLS OF THE TAMARINDUS INDICA SEED POLYSACCHARIDE: VISUAL EVALUATION BY MEANS OF INORGANIC AND ORGANIC MARKERS

    Directory of Open Access Journals (Sweden)

    P.C. Braga*, M. Dal Sasso, M. Culici

    2012-06-01

    Full Text Available The aim of this study was to investigate the possibility of using inorganic and organic markers to visualize the ability of the transparent polysaccharide (TSP polymer isolated from the endosperm of the seed kernel of Tamarindus indica, a tree that mainly grows in India and South-East Asia, to bind to human mucosal cells. A layer of human buccal cells was prepared on slides and overlaid by 0.2 ml of 0.6, 0.3, 0.15 and 0.075 % TSP solutions in phosphate buffer and then colloidal carbon black particles were deposited on the slides. The unbound colloidal carbon black particles were cleared by thoroughly washing the slides. The slides were then examined by means of Nomarski interference contrast microscopy in order to visualize the degree of surface retention of the black particles by the buccal cells. The same procedure was followed using Escherichia coli as organic markers. The clearly visible binding of black carbon particles to the cells treated with polymer revealed the presence of a thin layer of TSP covering the cells (untreated cells had no black carbon particles binding. The presence of the TSP has also been confirmed by a significant reduction in bacterial adhesiveness. Both markers made it possible to visualize the binding of the thin transparent layer of TSP and its retention, which was proportional to the degree of dilution. Using Escherichia coli it has been observed the possibility of counteracting the lock-and-key mechanism of micro-organism adhesion using the bioadhesive properties of this polymer to prevent possible contact between microorganism adhesins and complementary receptors.

  5. Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing.

    Science.gov (United States)

    Wolff, Bernard J; Benitez, Alvaro J; Desai, Heta P; Morrison, Shatavia S; Diaz, Maureen H; Winchell, Jonas M

    2017-03-01

    We developed a multiplex real-time PCR assay for simultaneously detecting M. pneumoniae and typing into historically-defined P1 types. Typing was achieved based on the presence of short type-specific indels identified through whole genome sequencing. This assay was 100% specific compared to existing methods and may be useful during epidemiologic investigations.

  6. Visualization Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — FUNCTION: Evaluates and improves the operational effectiveness of existing and emerging electronic warfare systems. By analyzing and visualizing simulation results...

  7. Traffic Visualization

    DEFF Research Database (Denmark)

    Picozzi, Matteo; Verdezoto, Nervo; Pouke, Matti;

    2013-01-01

    In this paper, we present a space-time visualization to provide city's decision-makers the ability to analyse and uncover important "city events" in an understandable manner for city planning activities. An interactive Web mashup visualization is presented that integrates several visualization...... techniques to give a rapid overview of traffic data. We illustrate our approach as a case study for traffic visualization systems, using datasets from the city of Oulu that can be extended to other city planning activities. We also report the feedback of real users (traffic management employees, traffic police...

  8. A neural network model on self-organizing emergence of simple-cell receptive field with orientation selectivity in visual cortex

    Institute of Scientific and Technical Information of China (English)

    杨谦; 齐翔林; 汪云九

    2001-01-01

    In order to probe into the self-organizing emergence of simple cell orientation selectivity,we tried to construct a neural network model that consists of LGN neurons and simple cells in visual cortex and obeys the Hebbian learning rule. We investigated the neural coding and representation of simple cells to a natural image by means of this model. The results show that the structures of their receptive fields are determined by the preferred orientation selectivity of simple cells.However, they are also decided by the emergence of self-organization in the unsupervision learning process. This kind of orientation selectivity results from dynamic self-organization based on the interactions between LGN and cortex.

  9. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    Science.gov (United States)

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

  10. [application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

    Science.gov (United States)

    Shebanova, A S; Bogdanov, A G; Ismagulova, T T; Feofanov, A V; Semenyuk, P I; Muronets, V I; Erokhina, M V; Onishchenko, G E; Kirpichnikov, M P; Shaitan, K V

    2014-01-01

    This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

  11. Visual search

    NARCIS (Netherlands)

    Toet, A.; Bijl, P.

    2003-01-01

    Visual search, with or without the aid of optical or electro-optical instruments, plays a significant role in various types of military and civilian operations (e.g., reconnaissance, surveillance, and search and rescue). Advance knowledge of human visual search and target acquisition performance is

  12. Visual Thinking.

    Science.gov (United States)

    Arnheim, Rudolf

    Based on the more general principle that all thinking (including reasoning) is basically perceptual in nature, the author proposes that visual perception is not a passive recording of stimulus material but an active concern of the mind. He delineates the task of visually distinguishing changes in size, shape, and position and points out the…

  13. Real-time whole-body visualization of Chikungunya Virus infection and host interferon response in zebrafish.

    Directory of Open Access Journals (Sweden)

    Nuno Palha

    Full Text Available Chikungunya Virus (CHIKV, a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.

  14. Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam.

    Science.gov (United States)

    Liyanagunawardena, Nilukshi; Sivakumar, Thillaiampalam; Kothalawala, Hemal; Silva, Seekkuge Susil Priyantha; Battsetseg, Badgar; Lan, Dinh Thi Bich; Inoue, Noboru; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-01-01

    Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic

  15. Visual Impairment

    Science.gov (United States)

    ... What Causes Visual Impairment? People rarely lose their eyesight during their teen years. When they do, it's ... inflammation in the eye. It's often found in poor rural countries that have overcrowded living conditions and ...

  16. Visual cognition

    Energy Technology Data Exchange (ETDEWEB)

    Pinker, S.

    1985-01-01

    This book consists of essays covering issues in visual cognition presenting experimental techniques from cognitive psychology, methods of modeling cognitive processes on computers from artificial intelligence, and methods of studying brain organization from neuropsychology. Topics considered include: parts of recognition; visual routines; upward direction; mental rotation, and discrimination of left and right turns in maps; individual differences in mental imagery, computational analysis and the neurological basis of mental imagery: componental analysis.

  17. Type-specific human papillomavirus distribution in invasive cervical carcinomas in Paraguay. A study of 432 cases.

    Science.gov (United States)

    Kasamatsu, Elena; Cubilla, Antonio L; Alemany, Laia; Chaux, Alcides; Tous, Sara; Mendoza, Laura; Paez, Malvina; Klaustermeier, Jo Ellen; Quint, Wim; Lloveras, Belen; de Sanjose, Silvia; Muñoz, Nubia; Bosch, Francisco Xavier

    2012-10-01

    Cervical carcinoma is the most common malignant tumor among woman in Paraguay. Cytological screening programs have not been successful and a plan for human papillomavirus (HPV) based-screening program and/or vaccination is under evaluation. This study aimed to identify the contribution of HPV genotypes in invasive cervical cancer in Paraguay to provide essential background data to guide and assess the introduction and impact of new preventive strategies based on HPV. Four hundred thirty two histologically confirmed cases (1960-2004) were analyzed. HPV detection in paraffin blocks was performed at the Catalan Institute of Oncology using PCR with SPF-10 broad spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe analysis. The majority of cases were squamous cell carcinoma (92.8%). Mean patients age was 48 years old. HPV DNA was detected in 73.1% of the cases and single infections were predominant (97.8%). The most common HPV single types were 16, 18, 45, 33, 31, 52, 35, and 39. 73.1% of HPV positive cases had an HPV 16, 18 as single infection. HPV16 was frequent in SCC whereas HPV 18 and 45 were prevalent in glandular tumors. Significant decrease of HPV 16 with age groups (P-trend = 0.022) and increase in other HPV types (P-trend > 0.001) were observed. The potential impact of HPV 16 and 18 for a vaccination program was 73.1%. The study provide a profile of the HPV situation in the country, with robust clinical, pathological and virological data which would permit a better cervical cancer screening and vaccination programs.

  18. PopulationProfiler: A Tool for Population Analysis and Visualization of Image-Based Cell Screening Data

    OpenAIRE

    Matuszewski, Damian J.; Carolina Wählby; Jordi Carreras Puigvert; Ida-Maria Sintorn

    2016-01-01

    Image-based screening typically produces quantitative measurements of cell appearance. Large-scale screens involving tens of thousands of images, each containing hundreds of cells described by hundreds of measurements, result in overwhelming amounts of data. Reducing per-cell measurements to the averages across the image(s) for each treatment leads to loss of potentially valuable information on population variability. We present PopulationProfiler-a new software tool that reduces per-cell mea...

  19. Visual comparison for information visualization

    KAUST Repository

    Gleicher, M.

    2011-09-07

    Data analysis often involves the comparison of complex objects. With the ever increasing amounts and complexity of data, the demand for systems to help with these comparisons is also growing. Increasingly, information visualization tools support such comparisons explicitly, beyond simply allowing a viewer to examine each object individually. In this paper, we argue that the design of information visualizations of complex objects can, and should, be studied in general, that is independently of what those objects are. As a first step in developing this general understanding of comparison, we propose a general taxonomy of visual designs for comparison that groups designs into three basic categories, which can be combined. To clarify the taxonomy and validate its completeness, we provide a survey of work in information visualization related to comparison. Although we find a great diversity of systems and approaches, we see that all designs are assembled from the building blocks of juxtaposition, superposition and explicit encodings. This initial exploration shows the power of our model, and suggests future challenges in developing a general understanding of comparative visualization and facilitating the development of more comparative visualization tools. © The Author(s) 2011.

  20. Postnatal Dendritic Growth and Spinogenesis of Layer-V Pyramidal Cells Differ between Visual, Inferotemporal, and Prefrontal Cortex of the Macaque Monkey

    Science.gov (United States)

    Oga, Tomofumi; Elston, Guy N.; Fujita, Ichiro

    2017-01-01

    Pyramidal cells in the primate cerebral cortex, particularly those in layer III, exhibit regional variation in both the time course and magnitude of postnatal growth and pruning of dendrites and spines. Less is known about the development of pyramidal cell dendrites and spines in other cortical layers. Here we studied dendritic morphology of layer-V pyramidal cells in primary visual cortex (V1, sensory), cytoarchitectonic area TE in the inferotemporal cortex (sensory association), and granular prefrontal cortex (Walker's area 12, executive) of macaque monkeys at the ages of 2 days, 3 weeks, 3.5 months, and 4.5 years. We found that changes in the basal dendritic field area of pyramidal cells were different across the three areas. In V1, field size became smaller over time (largest at 2 days, half that size at 4.5 years), in TE it did not change, and in area 12 it became larger over time (smallest at 2 days, 1.5 times greater at 4.5 years). In V1 and TE, the total number of branch points in the basal dendritic trees was similar between 2 days and 4.5 years, while in area 12 the number was greater in the adult monkeys than in the younger ones. Spine density peaked at 3 weeks and declined in all areas by adulthood, with V1 exhibiting a faster decline than area TE or area 12. Estimates of the total number of spines in the dendritic trees revealed that following the onset of visual experience, pyramidal cells in V1 lose more spines than they grow, whereas those in TE and area 12 grow more spines than they lose during the same period. These data provide further evidence that the process of synaptic refinement in cortical pyramidal cells differs not only according to time, but also location within the cortex. Furthermore, given the previous finding that layer-III pyramidal cells in all these areas exhibit the highest density and total number of spines at 3.5 months, the current results indicate that pyramidal cells in layers III and V develop spines at different rates.

  1. Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein

    Directory of Open Access Journals (Sweden)

    Bewley J Derek

    2008-09-01

    Full Text Available Abstract Background The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell. Results A novel, monomeric (m Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins. Conclusion The mEosFP::FABD-mTn probe retains

  2. Recovery of Visual Function in a Patient with an Onodi Cell Mucocele Compressive Optic Neuropathy Who Had a 5-Week Interval between Onset and Surgical Intervention: A Case Report

    Directory of Open Access Journals (Sweden)

    Wencan Wu

    2010-01-01

    Full Text Available Purpose. To report on a patient with compressive optic neuropathy secondary to an Onodi cell mucocele, who fully recovered visual function following surgery. Method. Case report. Results. A 28-year-old male was admitted with a right visual acuity of 20/100 following treatment for an initial diagnosis of optic neuritis. Subsequent examination suggested compressive optic neuropathy, and neuroimaging confirmed the presence of an Onodi mucocele compressing the optic nerve. The patient underwent a right endonasal sphenoethmoidectomy with decompression 5 weeks after the initial onset of symptoms. Three weeks following surgery, the visual acuity was 20/20, and there was complete resolution of the visual field defect, which has remained stable at 1 year. Conclusion. Onodi cell mucocele should be included in the differential diagnosis of a young patient with compressive optic neuropathy. Surgical decompression should be considered even when symptoms have been present for over a month.

  3. Visual Education

    DEFF Research Database (Denmark)

    Buhl, Mie; Flensborg, Ingelise

    2010-01-01

    The intrinsic breadth of various types of images creates new possibilities and challenges for visual education. The digital media have moved the boundaries between images and other kinds of modalities (e.g. writing, speech and sound) and have augmented the possibilities for integrating the functi......The intrinsic breadth of various types of images creates new possibilities and challenges for visual education. The digital media have moved the boundaries between images and other kinds of modalities (e.g. writing, speech and sound) and have augmented the possibilities for integrating...... the functions of the images and visuality in new forms of knowledge-building beyond traditional boundaries of school subjects. New learning cultures emerge and place the visual skills of decoding and meaning-making in the centre of future competences for citizens in a globalised world.   This paper discusses...... the learning potential of images and visuality from two perspectives: 1) The perspective of digital media which are assumed to form an increasing part of experience and communication from the use of internet, tv and mobile devices. 2) The perspective of culture where images and visualisations are assumed...

  4. Compass Cells in the Brain of an Insect Are Sensitive to Novel Events in the Visual World.

    Directory of Open Access Journals (Sweden)

    Tobias Bockhorst

    Full Text Available The central complex of the insect brain comprises a group of neuropils involved in spatial orientation and memory. In fruit flies it mediates place learning based on visual landmarks and houses neurons that encode the orientation for goal-directed locomotion, based on landmarks and self-motion cues for angular path-integration. In desert locusts, the central complex holds a compass-like representation of head directions, based on the polarization pattern of skylight. Through intracellular recordings from immobilized locusts, we investigated whether sky compass neurons of the central complex also represent the position or any salient feature of possible landmarks, in analogy to the observations in flies. Neurons showed strongest responses to the novel appearance of a small moving square, but we found no evidence for a topographic representation of object positions. Responses to an individual square were independent of direction of motion and trajectory, but showed rapid adaptation to successive stimulation, unaffected by changing the direction of motion. Responses reappeared, however, if the moving object changed its trajectory or if it suddenly reversed moving direction against the movement of similar objects that make up a coherent background-flow as induced by ego-motion. Response amplitudes co-varied with the precedent state of dynamic background activity, a phenomenon that has been related to attention-dependent saliency coding in neurons of the mammalian primary visual cortex. The data show that neurons of the central complex of the locust brain are visually bimodal, signaling sky compass direction and the novelty character of moving objects. These response properties might serve to attune compass-aided locomotor control to unexpected events in the environment. The difference to data obtained in fruit flies might relate to differences in the lifestyle of landmark learners (fly and compass navigators (locust, point to the existence of

  5. Visualizing Transformation

    DEFF Research Database (Denmark)

    Pedersen, Pia

    2012-01-01

    Transformation, defined as the step of extracting, arranging and simplifying data into visual form (M. Neurath, 1974), was developed in connection with ISOTYPE (International System Of TYpographic Picture Education) and might well be the most important legacy of Isotype to the field of graphic...... design. Recently transformation has attracted renewed interest because of the book ‘The Transformer’ written by Robin Kinross and Marie Neurath. My on-going research project, summarized in this paper, identifies and depicts the essential principles of data visualization underlying the process...... of transformation with reference to Marie Neurath’s sketches on the Bilston Project. The material has been collected at the Otto and Marie Neurath Collection housed at the University of Reading, UK. By using data visualization as a research method to look directly into the process of transformation the project...

  6. Visual cognition

    Energy Technology Data Exchange (ETDEWEB)

    Pinker, S.

    1985-01-01

    This collection of research papers on visual cognition first appeared as a special issue of Cognition: International Journal of Cognitive Science. The study of visual cognition has seen enormous progress in the past decade, bringing important advances in our understanding of shape perception, visual imagery, and mental maps. Many of these discoveries are the result of converging investigations in different areas, such as cognitive and perceptual psychology, artificial intelligence, and neuropsychology. This volume is intended to highlight a sample of work at the cutting edge of this research area for the benefit of students and researchers in a variety of disciplines. The tutorial introduction that begins the volume is designed to help the nonspecialist reader bridge the gap between the contemporary research reported here and earlier textbook introductions or literature reviews.

  7. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  8. Visualizing Cell Architecture and Molecular Location Using Soft X-Ray Tomography and Correlated Cryo-Light Microscopy

    Science.gov (United States)

    McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

    2012-05-01

    Living cells are structured to create a range of microenvironments that support specific chemical reactions and processes. Understanding how cells function therefore requires detailed knowledge of both the subcellular architecture and the location of specific molecules within this framework. Here we review the development of two correlated cellular imaging techniques that fulfill this need. Cells are first imaged using cryogenic fluorescence microscopy to determine the location of molecules of interest that have been labeled with fluorescent tags. The same specimen is then imaged using soft X-ray tomography to generate a high-contrast, 3D reconstruction of the cells. Data from the two modalities are then combined to produce a composite, information-rich view of the cell. This correlated imaging approach can be applied across the spectrum of problems encountered in cell biology, from basic research to biotechnological and biomedical applications such as the optimization of biofuels and the development of new pharmaceuticals.

  9. Loss of Responses to Visual But Not Electrical Stimulation in Ganglion Cells of Rats With Severe Photoreceptor Degeneration

    OpenAIRE

    2009-01-01

    Retinal implants are intended to help patients with degenerative conditions by electrically stimulating surviving cells to produce artificial vision. However, little is known about how individual retinal ganglion cells respond to direct electrical stimulation in degenerating retina. Here we used a transgenic rat model to characterize ganglion cell responses to light and electrical stimulation during photoreceptor degeneration. Retinas from pigmented P23H-1 rats were compared with wild-type re...

  10. Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging.

    Science.gov (United States)

    Miwa, Shinji; Yano, Shuya; Kimura, Hiroaki; Yamamoto, Mako; Toneri, Makoto; Murakami, Takashi; Hayashi, Katsuhiro; Yamamoto, Norio; Fujiwara, Toshiyoshi; Tsuchiya, Hiroyuki; Hoffman, Robert M

    2015-01-01

    The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m(2) UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G1/S transition were analyzed using the Kaplan-Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G2/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m(2) UVB during G0/G1 phase had a higher survival rate than the cells irradiated during S/G2/M phase. A minority of cells could escape S/G2/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G1/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m(2) resulted in a greater number of apoptotic cells.

  11. In vivo visualization and monitoring of viable neural stem cells using noninvasive bioluminescence imaging in the 6-hydroxydopamine-induced mouse model of Parkinson disease.

    Science.gov (United States)

    Im, Hyung-Jun; Hwang, Do Won; Lee, Han Kyu; Jang, Jaeho; Lee, Song; Youn, Hyewon; Jin, Yeona; Kim, Seung U; Kim, E Edmund; Kim, Yong Sik; Lee, Dong Soo

    2013-06-01

    Transplantation of neural stem cells (NSCs) has been proposed as a treatment for Parkinson disease (PD). The aim of this study was to monitor the viability of transplanted NSCs expressing the enhanced luciferase gene in a mouse model of PD in vivo. The PD animal model was induced by unilateral injection of 6-hydroxydopamine (6-OHDA). The behavioral test using apomorphine-induced rotation and positron emission tomography with [18F]N-(3-fluoropropyl)-2'-carbomethoxy-3'-(4-iodophenyl)nortropane ([18F]FP-CIT) were conducted. HB1.F3 cells transduced with an enhanced firefly luciferase retroviral vector (F3-effLuc cells) were transplanted into the right striatum. In vivo bioluminescence imaging was repeated for 2 weeks. Four weeks after transplantation, [18F]FP-CIT PET and the rotation test were repeated. All 6-OHDA-injected mice showed markedly decreased [18F]FP-CIT uptake in the right striatum. Transplanted F3-effLuc cells were visualized on the right side of the brain in all mice by bioluminescence imaging. The bioluminescence intensity of the transplanted F3-effLuc cells gradually decreased until it was undetectable by 10 days. The behavioral test showed that stem cell transplantation attenuated the motor symptoms of PD. No significant change was found in [18F]FP-CIT imaging after cell transplantation. We successfully established an in vivo bioluminescence imaging system for the detection of transplanted NSCs in a mouse model of PD. NSC transplantation induced behavioral improvement in PD model mice.

  12. Visualizing inequality

    Science.gov (United States)

    Eliazar, Iddo

    2016-07-01

    The study of socioeconomic inequality is of substantial importance, scientific and general alike. The graphic visualization of inequality is commonly conveyed by Lorenz curves. While Lorenz curves are a highly effective statistical tool for quantifying the distribution of wealth in human societies, they are less effective a tool for the visual depiction of socioeconomic inequality. This paper introduces an alternative to Lorenz curves-the hill curves. On the one hand, the hill curves are a potent scientific tool: they provide detailed scans of the rich-poor gaps in human societies under consideration, and are capable of accommodating infinitely many degrees of freedom. On the other hand, the hill curves are a powerful infographic tool: they visualize inequality in a most vivid and tangible way, with no quantitative skills that are required in order to grasp the visualization. The application of hill curves extends far beyond socioeconomic inequality. Indeed, the hill curves are highly effective 'hyperspectral' measures of statistical variability that are applicable in the context of size distributions at large. This paper establishes the notion of hill curves, analyzes them, and describes their application in the context of general size distributions.

  13. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-03-17

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

  14. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  15. Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics

    Directory of Open Access Journals (Sweden)

    Silver Pamela A

    2006-04-01

    Full Text Available Abstract Background Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. Results We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. Conclusion Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to

  16. Tissue-type-specific heat-shock response and immunolocalization of class I low-molecular-weight heat-shock proteins in soybean

    Energy Technology Data Exchange (ETDEWEB)

    Tsung-Luo Jinn; Pi-Fang Linda Chang; Yih-Ming Chen [National Taiwan Univ. (China)] [and others

    1997-06-01

    A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soy-bean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40{degrees}C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40{degrees}C {yields} 28{degrees}C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.

  17. Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Eijiro Yamada

    2012-05-01

    Full Text Available Skeletal muscle atrophy induced by aging (sarcopenia, inactivity, and prolonged fasting states (starvation is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

  18. Canine mesenchymal stem cells are effectively labeled with silica nanoparticles and unambiguously visualized in highly autofluorescent tissues

    Directory of Open Access Journals (Sweden)

    Han Sei-Myoung

    2012-08-01

    Full Text Available Abstract Background Development of a method for long-term labeling of cells is critical to elucidate transplanted cell fate and migration as well as the contribution to tissue regeneration. Silica nanoparticles have been recently developed and demonstrated to be biocompatible with a high labeling capacity. Thus, our study was designed to assess the suitability of silica nanoparticles for labeling canine mesenchymal stem cells (MSCs and the fluorescence afficiency in highly autofluorescent tissue. Results We examined the effect of silica nanoparticle labeling on stem cell morphology, viability and differentiation as compared with those of unlabeled control cells. After 4 h of incubation with silica nanoparticles, they were internalized by canine MSCs without a change in the morphology of cells compared with that of control cells. The viability and proliferation of MSCs labeled with silica nanoparticles were evaluated by a WST-1 assay and trypan blue exclusion. No effects on cell viability were observed, and the proliferation of canine MSCs was not inhibited during culture with silica nanoparticles. Furthermore, adipogenic and osteogenic differentiation of silica nanoparticle-labeled canine MSCs was at a similar level compared with that of unlabeled cells, indicating that silica nanoparticle labeling did not alter the differentiation capacity of canine MSCs. Silica nanoparticle-labeled canine MSCs were injected into the kidneys of BALB/c mice after celiotomy, and then the mice were sacrificed after 2 or 3 weeks. The localization of injected MSCs was closely examined in highly autofluorescent renal tissues. Histologically, canine MSCs were uniformly and completely labeled with silica nanoparticles, and were unambiguously imaged in histological sections. Conclusions The results of the current study showed that silica nanoparticles are useful as an effective labeling marker for MSCs, which can elucidate the distribution and fate of transplanted

  19. Workflow for the integration of a realistic 3D geomodel in process simulations using different cell types and advanced scientific visualization: Variations on a synthetic salt diapir

    Science.gov (United States)

    Görz, Ines; Herbst, Martin; Börner, Jana H.; Zehner, Björn

    2017-03-01

    The purpose of this study is to use one complex geological 3D model for numerical simulations of various physical processes in process-specific simulation software. To do this, the 3D model has to be discretized according to different cell types, depending on the requirements of the simulation method. We used a salt structure with a diapir and its deformed host rock to produce two 3D models describing the boundary surfaces of the structure: one very simplified model consisting of cuboid surfaces and a realistic model consisting of irregular boundary surfaces. We provide a workflow for how to generate hexahedral, tetrahedral and spherical volume representations of these two geometries. We utilized the volume representations to simulate temperature, displacement and transient electromagnetic fields. We can show that the simulation results closely reflect the input geometry and that it is worth the effort to produce geometric models that are as realistic as possible. Additionally, we provide a workflow for simultaneous visualization and analysis of the simulation results. Scientific visualization is an important tool for deriving knowledge from complex investigations.

  20. NSOM/QD-Based Visualization of GM1 Serving as Platforms for TCR/CD3 Mediated T-Cell Activation

    Directory of Open Access Journals (Sweden)

    Liyun Zhong

    2013-01-01

    Full Text Available Direct molecular imaging of nanoscale relationship between T-cell receptor complexes (TCR/CD3 and gangliosidosis GM1 before and after T-cell activation has not been reported. In this study, we made use of our expertise of near-field scanning optical microscopy(NSOM/immune-labeling quantum dots- (QD-based dual-color imaging system to visualize nanoscale profiles for distribution and organization of TCR/CD3, GM1, as well as their nanospatial relationship and their correlation with PKCθ signaling cascade during T-cell activation. Interestingly, after anti-CD3/anti-CD28 Ab co-stimulation, both TCR/CD3 and GM1 were clustered to form nanodomains; moreover, all of TCR/CD3 nanodomains were colocalized with GM1 nanodomains, indicating that the formation of GM1 nanodomains was greatly correlated with TCR/CD3 mediated signaling. Specially, while T-cells were pretreated with PKCθ signaling inhibitor rottlerin to suppress IL-2 cytokine production, no visible TCR/CD3 nanodomains appeared while a lot of GM1 nanodomains were still observed. However, while T-cells are pretreated with PKCαβ signaling inhibitor GÖ6976 to suppress calcium-dependent manner, all of TCR/CD3 nanodomains were still colocalized with GM1 nanodomains. These findings possibly support the notion that the formation of GM1 nanodomains indeed serves as platforms for the recruitment of TCR/CD3 nanodomains, and TCR/CD3 nanodomains are required for PKCθ signaling cascades and T-cell activation

  1. TLM-Quant : An Open-Source Pipeline for Visualization and Quantification of Gene Expression Heterogeneity in Growing Microbial Cells

    NARCIS (Netherlands)

    Piersma, Sjouke; Denham, Emma L.; Drulhe, Samuel; Tonk, Rudi H. J.; Schwikowski, Benno; van Dijl, Jan Maarten

    2013-01-01

    Gene expression heterogeneity is a key driver for microbial adaptation to fluctuating environmental conditions, cell differentiation and the evolution of species. This phenomenon has therefore enormous implications, not only for life in general, but also for biotechnological applications where unwan

  2. Aged human bone marrow stromal cells maintaining bone forming capacity in vivo evaluated using an improved method of visualization

    DEFF Research Database (Denmark)

    Stenderup, Karin; Rosada, Cecilia; Justesen, J;

    2004-01-01

    an in vivo assay for quantifying the bone forming capacity (BFC) and we compared the BFC of osteoblastic cells obtained from young and old donors. Osteoblasts were obtained from human bone marrow stromal cell cultures and implanted subcutaneously in immuno-deficient mice (NOD/LtSz- Prkdc(scid)). After 8...... weeks, the implants were removed and embedded un-decalcified in methyl methacrylate (MMA). Sections were stained histochemically with Goldner's Trichrome stain and immuno-histochemically using human-specific antibodies against known osteogenic markers. Implanted human marrow stromal cells (hMSC) were...... able to form bone in vivo. The donor origin of bone was verified using several human-specific antibodies. Dose-response experiments demonstrated that 5 x 10(5) hMSC per implant gave the maximal bone formation after 8 weeks. No difference in BFC was observed between cells obtained from young (24...

  3. Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

    Science.gov (United States)

    Ginart, Paul; Kalish, Jennifer M; Jiang, Connie L; Yu, Alice C; Bartolomei, Marisa S; Raj, Arjun

    2016-03-01

    Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

  4. Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells.

    Science.gov (United States)

    Moats, R K; Ramirez, V D

    2000-09-01

    Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17beta-diol 6-(O-carboxymethyl)oxime:(125)I-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17beta-diol 17-hemisuccinate:( 125)I-BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA. This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast, (125! )I-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multivesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min post-exposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. Labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17beta-estradiol, but not 17alpha-E6 BSA, 17alpha-estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first t! o visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures

  5. Cell-Type Specific Channelopathies in the Prefrontal Cortex of the fmr1-/y Mouse Model of Fragile X Syndrome 1,2,3

    OpenAIRE

    Kalmbach, Brian E.; Johnston, Daniel; Brager, Darrin H.

    2015-01-01

    Abstract Fragile X syndrome (FXS) is caused by transcriptional silencing of the fmr1 gene resulting in the loss of fragile X mental retardation protein (FMRP) expression. FXS patients display several behavioral phenotypes associated with prefrontal cortex (PFC) dysfunction. Voltage-gated ion channels, some of which are regulated by FMRP, heavily influence PFC neuron function. Although there is evidence for brain region-specific alterations to the function a single type of ion channel in FXS, ...

  6. Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity

    NARCIS (Netherlands)

    Hrdlickova, Barbara; Kumar, Vinod; Kanduri, Kartiek; Zhernakova, Daria V.; Tripathi, Subhash; Karjalainen, Juha; Lund, Riikka J.; Li, Yang; Ullah, Ubaid; Modderman, Rutger; Abdulahad, Wayel; Lahdesmaki, Harri; Franke, Lude; Lahesmaa, Riitta; Wijmenga, Cisca; Withoff, Sebo

    2014-01-01

    Background: Although genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding

  7. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Directory of Open Access Journals (Sweden)

    Yu-Yo Sun

    Full Text Available The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α through inhibition of prolyl hydrolase 2 (PHD2 and activation of the phosphatidylinositide-3 kinase (PI3K/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo and vascular endothelial growth factor (VEGF, two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  8. Cell type-specific dependency on the PI3K/Akt signaling pathway for the endogenous Epo and VEGF induction by baicalein in neurons versus astrocytes.

    Science.gov (United States)

    Sun, Yu-Yo; Lin, Shang-Hsuan; Lin, Hung-Cheng; Hung, Chia-Chi; Wang, Chen-Yu; Lin, Yen-Chu; Hung, Kuo-Sheng; Lien, Cheng-Chang; Kuan, Chia-Yi; Lee, Yi-Hsuan

    2013-01-01

    The neuroprotective effect of baicalein is generally attributed to inhibition of 12/15-lipoxygenase (12/15-LOX) and suppression of oxidative stress, but recent studies showed that baicalein also activates hypoxia-inducible factor-α (HIF1α) through inhibition of prolyl hydrolase 2 (PHD2) and activation of the phosphatidylinositide-3 kinase (PI3K)/Akt signaling pathway. Yet, the significance and regulation of prosurvival cytokines erythropoietin (Epo) and vascular endothelial growth factor (VEGF), two transcriptional targets of HIF1α, in baicalein-mediated neuroprotection in neurons and astrocytes remains unknown. Here we investigated the causal relationship between the PI3K/Akt signaling pathway and Epo/VEGF expression in baicalein-mediated neuroprotection in primary rat cortical neurons and astrocytes. Our results show that baicalein induced Epo and VEGF expression in a HIF1α- and PI3K/Akt-dependent manner in neurons. Baicalein also protected neurons against excitotoxicity in a PI3K- and Epo/VEGF-dependent manner without affecting neuronal excitability. In contrast, at least a 10-fold higher concentration of baicalein was needed to induce Epo/VEGF production and PI3K/Akt activity in astrocytes for protection of neurons. Moreover, only baicalein-induced astrocytic VEGF, but not Epo expression requires HIF1α, while PI3K/Akt signaling had little role in baicalein-induced astrocytic Epo/VEGF expression. These results suggest distinct mechanisms of baicalein-mediated Epo/VEGF production in neurons and astrocytes for neuroprotection, and provide new insights into the mechanisms and potential of baicalein in treating brain injury in vivo.

  9. Antiestrogenic activity of flavnoid phytochemicals mediated via c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha

    Science.gov (United States)

    Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling in...

  10. Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

    DEFF Research Database (Denmark)

    Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard;

    2008-01-01

    The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators...

  11. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

    Science.gov (United States)

    Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

    2013-03-15

    Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.

  12. Chronic treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside increases insulin-stimulated glucose uptake and GLUT4 translocation in rat skeletal muscles in a fiber type-specific manner.

    Science.gov (United States)

    Buhl, E S; Jessen, N; Schmitz, O; Pedersen, S B; Pedersen, O; Holman, G D; Lund, S

    2001-01-01

    Recent studies have demonstrated that chronic administration of AICAR (5-aminoimidazole-4-carboxamide- 1-beta-D-ribofuranoside), an activator of the AMP-activated protein kinase, increases hexokinase activity and the contents of total GLUT4 and glycogen in rat skeletal muscles. To explore whether AICAR also affects insulin-stimulated glucose transport and GLUT4 cell surface content, Wistar rats were subcutaneously injected with AICAR for 5 days in succession (1 mg/g body wt). Maximally insulin-stimulated (60 nmol/l) glucose uptake was markedly increased in epitrochlearis (EPI) muscle (average 63%, P fiber type-specific increase in insulin-stimulated glucose uptake and GLUT4 cell surface content in rat skeletal muscle with the greatest effect observed on white fast-twitch glycolytic muscles (EPI). These results are comparable with the effects of chronic exercise training, and it brings the AMP-activated protein kinase into focus as a new interesting target for future pharmacological intervention in insulin-resistant conditions.

  13. Identification and visualization of CD8+ T cell mediated IFN-γ signaling in target cells during an antiviral immune response in the brain.

    Directory of Open Access Journals (Sweden)

    Mariana Puntel

    Full Text Available CD8(+ T cells infiltrate the brain during an anti-viral immune response. Within the brain CD8(+ T cells recognize cells expressing target antigens, become activated, and secrete IFNγ. However, there are no methods to recognize individual cells that respond to IFNγ. Using a model that studies the effects of the systemic anti-adenoviral immune response upon brain cells infected with an adenoviral vector in mice, we describe a method that identifies individual cells that respond to IFNγ. To identify individual mouse brain cells that respond to IFNγ we constructed a series of adenoviral vectors that contain a transcriptional response element that is selectively activated by IFNγ signaling, the gamma-activated site (GAS promoter element; the GAS element drives expression of a transgene, Cre recombinase (Ad-GAS-Cre. Upon binding of IFNγ to its receptor, the intracellular signaling cascade activates the GAS promoter, which drives expression of the transgene Cre recombinase. We demonstrate that upon activation of a systemic immune response against adenovirus, CD8(+ T cells infiltrate the brain, interact with target cells, and cause an increase in the number of cells expressing Cre recombinase. This method can be used to identify, study, and eventually determine the long term fate of infected brain cells that are specifically targeted by IFNγ. The significance of this method is that it will allow to characterize the networks in the brain that respond to the specific secretion of IFNγ by anti-viral CD8(+ T cells that infiltrate the brain. This will allow novel insights into the cellular and molecular responses underlying brain immune responses.

  14. Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography.

    Science.gov (United States)

    Darrow, Michele C; Zhang, Yujin; Cinquin, Bertrand P; Smith, Elizabeth A; Boudreau, Rosanne; Rochat, Ryan H; Schmid, Michael F; Xia, Yang; Larabell, Carolyn A; Chiu, Wah

    2016-09-15

    Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

  15. Transcriptional analysis of rat photoreceptor cells reveals daily regulation of genes important for visual signaling and light damage susceptibility.

    Science.gov (United States)

    Kunst, Stefanie; Wolloscheck, Tanja; Hölter, Philip; Wengert, Alexander; Grether, Markus; Sticht, Carsten; Weyer, Veronika; Wolfrum, Uwe; Spessert, Rainer

    2013-03-01

    Photoreceptor cells face the challenge of adjusting their function and, possibly, their susceptibility to light damage to the marked daily changes in ambient light intensity. To achieve a better understanding of photoreceptor adaptation at the transcriptional level, this study aimed to identify genes which are under daily regulation in photoreceptor cells using microarray analysis and quantitative PCR. Included in the gene set obtained were a number of genes which up until now have not been shown to be expressed in photoreceptor cells, such as Atf3 (activating transcription factor 3) and Pde8a (phosphodiesterase 8A), and others with a known impact on phototransduction and/or photoreceptor survival, such as Grk1 (G protein-coupled receptor kinase 1) and Pgc-1α (peroxisome proliferator-activated receptor γ, coactivator 1alpha). According to their daily dynamics, the genes identified could be clustered in two groups: those with peak expression during the second part of the day which are uniformly promoted to cycle by light/dark transitions and those with peak expression during the second part of the night which are predominantly driven by a clock. Since Grk1 and Pgc-1α belong in the first group, the present results support a concept in which transcriptional regulation of genes by ambient light contributes to the functional adjustment of photoreceptor cells over the 24-h period.

  16. Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan); Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)

    2012-06-01

    Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

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

    Science.gov (United States)

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

    2012-01-15

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

  18. Correlation networks visualization

    Directory of Open Access Journals (Sweden)

    Nicholas J. Provart

    2012-10-01

    Full Text Available New, in silico ways of generating hypotheses based on large data sets have emerged in the past decade. These data sets have been used to investigate different aspects of plant biology, especially at the level of transcriptome, from tissue-specific expression patterns to patterns in as little as a few cells. Such publicly-available data are a boon to researchers for hypothesis generation by providing a guide for experimental work such as phenotyping or genetic analysis. More advanced computational methods can leverage these data via gene coexpression analysis, the results of which can be visualized and refined using network analysis. Other kinds of networks of e.g. protein-protein interactions, can also be used to inform biology. These networks can be visualized and analyzed with additional information on gene expression levels, subcellular localization, etc., or with other emerging kinds information. Finally, cross-level correlation is an area that will become increasingly important. Visualizing these cross-level correlations will require new data visualization tools.

  19. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system.

    Science.gov (United States)

    Anton, Tobias; Bultmann, Sebastian; Leonhardt, Heinrich; Markaki, Yolanda

    2014-01-01

    Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture.

  20. Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease.

    Science.gov (United States)

    Cortese-Krott, Miriam M; Rodriguez-Mateos, Ana; Sansone, Roberto; Kuhnle, Gunter G C; Thasian-Sivarajah, Sivatharsini; Krenz, Thomas; Horn, Patrick; Krisp, Christoph; Wolters, Dirk; Heiß, Christian; Kröncke, Klaus-Dietrich; Hogg, Neil; Feelisch, Martin; Kelm, Malte

    2012-11-15

    A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.

  1. Renewable bio ionic liquids-water mixtures-mediated selective removal of lignin from rice straw: visualization of changes in composition and cell wall structure.

    Science.gov (United States)

    Hou, Xue-Dan; Li, Ning; Zong, Min-Hua

    2013-07-01

    Pretreatment of rice straw by using renewable cholinium amino acids ionic liquids ([Ch][AA] ILs)-water mixtures and the subsequent enzymatic hydrolysis of the residues were conducted in the present work. Of the eight mixtures composed of ILs and water, most were found to be effective for rice straw pretreatment. After pretreatment with 50% ILs-water mixtures, the enzymatic digestion of the lignocellulosic biomass was enhanced significantly, thus leading to satisfactory sugar yields of >80% for glucose and approximately 50% for xylose. To better understand the ILs pretreatment mechanism, confocal laser scanning microscopy combined with immunolabeling and transmission electron microscopy were used to visualize changes in the contents and distribution of two major components--lignin and xylan. The results coupled with changes in chemical structures (infrared spectra) of the substrates indicated occurrence of extensive delignification, especially in cell corner and compound middle lumen of cell walls, which made polysaccharides more accessible to enzymes. This pretreatment process is promising for large-scale application because of the high sugar yields, easy handling, being environmentally benign and highly tolerant to moisture, and significantly reduced cost and energy consumption.

  2. Glow in the Dark: Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants

    Institute of Scientific and Technical Information of China (English)

    Wenzislava Ckurshumova; Adriana E. Caragea; Rochelle S. Goldstein; Thomas Berleth

    2011-01-01

    Since the hallmark discovery of Aequorea victoria's Green Fluorescent Protein (GFP) and its adaptation for efficient use in plants,fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types,to monitor dynamic cell fate selection processes,and to obtain cell type-specific transcriptomes.Fluorescent tagging enabled visualization in living tissues and the precise recordings of dynamic expression pattern changes.The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms.In developmental studies,the use of fluorescent proteins has become critical,where morphological markers of tissues,cell types,or differentiation stages are either not known or not easily recognizable.In this review,we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

  3. Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells.

    Directory of Open Access Journals (Sweden)

    Le Tran Phuc Khoa

    Full Text Available Fibroblast growth factor 5 (Fgf5 has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.

  4. Bimolecular Complementation to Visualize Filovirus VP40-Host Complexes in Live Mammalian Cells: Toward the Identification of Budding Inhibitors

    Directory of Open Access Journals (Sweden)

    Yuliang Liu

    2011-01-01

    Full Text Available Virus-host interactions play key roles in promoting efficient egress of many RNA viruses, including Ebola virus (EBOV or “e” and Marburg virus (MARV or “m”. Late- (L- domains conserved in viral matrix proteins recruit specific host proteins, such as Tsg101 and Nedd4, to facilitate the budding process. These interactions serve as attractive targets for the development of broad-spectrum budding inhibitors. A major gap still exists in our understanding of the mechanism of filovirus budding due to the difficulty in detecting virus-host complexes and mapping their trafficking patterns in the natural environment of the cell. To address this gap, we used a bimolecular complementation (BiMC approach to detect, localize, and follow the trafficking patterns of eVP40-Tsg101 complexes in live mammalian cells. In addition, we used the BiMC approach along with a VLP budding assay to test small molecule inhibitors identified by in silico screening for their ability to block eVP40 PTAP-mediated interactions with Tsg101 and subsequent budding of eVP40 VLPs. We demonstrated the potential broad spectrum activity of a lead candidate inhibitor by demonstrating its ability to block PTAP-dependent binding of HIV-1 Gag to Tsg101 and subsequent egress of HIV-1 Gag VLPs.

  5. Visualization of in Vivo Hydrogen Sulfide Production by a Bioluminescence Probe in Cancer Cells and Nude Mice.

    Science.gov (United States)

    Tian, Xiaodong; Li, Zhiyan; Lau, Choiwan; Lu, Jianzhong

    2015-11-17

    Hydrogen sulfide (H2S) has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon monoxide. However, its precise measurement in living cells and animals remains a challenge. In this study, a novel bioluminescence H2S probe was designed and synthesized by modifying the 6'-amino group of d-aminoluciferin into a 6'-azido group, which was highly selective against other reactive sulfur, nitrogen, and oxygen species. Our H2S probe azidoluciferin sensitively reacted with H2S to release d-aminoluciferin with a strong bioluminescence signal. On the basis of its high selectivity and sensitivity, the H2S probe was used to detect H2S production in live cancer cells and nude mice. The bioluminescence signal decreased in mice treated with propargylglycine, an inhibitor of H2S, suggesting that our H2S probe can detect endogenous H2S in real time, in vivo. Overall, the excellent sensing properties of the probe combined with its bioimaging capability make it a useful tool to study H2S biological roles.

  6. Visual-servoing optical microscopy

    Science.gov (United States)

    Callahan, Daniel E.; Parvin, Bahram

    2009-06-09

    The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time: quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

  7. A visual thalamocortical slice.

    Science.gov (United States)

    MacLean, Jason N; Fenstermaker, Vivian; Watson, Brendon O; Yuste, Rafael

    2006-02-01

    We describe a thalamocortical slice preparation in which connectivity between the mouse lateral geniculate nucleus (LGN) and primary visual cortex (V1) is preserved. Through DiI injections in fixed brains we traced and created a three-dimensional model of the mouse visual pathways. From this computer model we designed a slice preparation that contains a projection from LGN to V1. We prepared brain slices with these predicted coordinates and demonstrated anatomical LGN-V1 connectivity in these slices after LGN tracer injections. We also revealed functional LGN-V1 connectivity by stimulating LGN electrically and detecting responses in layer 4 of V1 using calcium imaging, field potential recordings and whole-cell recordings. We also identified layer-4 neurons that receive direct thalamocortical input. Finally, we compared cortical activity after LGN stimulation with spontaneous cortical activity and found significant overlap of the spatiotemporal dynamics generated by both types of events.

  8. Type-specific HPV prevalence in cervical cancer and high-grade lesions in Latin America and the Caribbean: systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Agustín Ciapponi

    Full Text Available BACKGROUND: Cervical cancer is a major public health problem in Latin America and the Caribbean (LA&C, showing some of the highest incidence and mortality rates worldwide. Information on HPV type distribution in high-grade cervical lesions (HSIL and invasive cervical cancer (ICC is crucial to predict the future impact of HPV16/18 vaccines and screening programmes, and to establish an appropriate post-vaccinal virologic surveillance. The aim was to assess the prevalence of HPV types in HSIL and ICC in studies in LA&C. METHODS AND FINDINGS: We performed a systematic review, following the MOOSE guidelines for systematic reviews of observational studies, and the PRISMA statement for reporting systematic reviews and meta-analyses. Inclusion criteria were at least ten cases of HSIL/ICC, and HPV-type elicitation. The search, without language restrictions, was performed in MEDLINE, Cochrane Library, EMBASE, LILACS from inception date to December 2009, proceedings, reference lists and consulting experts. A meta-analysis was performed using arc-sine transformations to stabilize the variance of simple proportions. Seventy-nine studies from 18 countries were identified, including 2446 cases of HSIL and 5540 of ICC. Overall, 46.5% of HSIL cases harbored HPV 16 and 8.9% HPV18; in ICC, 53.2% of cases harbored HPV 16 and 13.2% HPV 18. The next five most common types, in decreasing frequency, were HPV 31, 58, 33, 45, and 52. Study's limitations comprise the cross-sectional design of most included studies and their inherent risk of bias, the lack of representativeness, and variations in the HPV type-specific sensitivity of different PCR protocols. CONCLUSIONS: This study is the broadest summary of HPV type distribution in HSIL and ICC in LA&C to date. These data are essential for local decision makers regarding HPV screening and vaccination policies. Continued HPV surveillance would be useful, to assess the potential for changing type-specific HPV prevalence in

  9. Single-tube multiplex PCR using type-specific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia

    Directory of Open Access Journals (Sweden)

    Warenholt Janina

    2011-01-01

    Full Text Available Abstract Background Human papillomavirus (HPV E6/E7 type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set. A single-tube multiplex PCR containing type-specific primers was developed to target the E6/E7 genes of two low-risk and 19 high-risk genotypes (HPV6, 11 and 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82 and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis. Results The method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA Microcards® imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above, except one adenocarcinoma, contained E6/E7 genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore E6/E7 DNA; all but one were HPV16. One neck

  10. Visualization rhetoric: framing effects in narrative visualization.

    Science.gov (United States)

    Hullman, Jessica; Diakopoulos, Nicholas

    2011-12-01

    Narrative visualizations combine conventions of communicative and exploratory information visualization to convey an intended story. We demonstrate visualization rhetoric as an analytical framework for understanding how design techniques that prioritize particular interpretations in visualizations that "tell a story" can significantly affect end-user interpretation. We draw a parallel between narrative visualization interpretation and evidence from framing studies in political messaging, decision-making, and literary studies. Devices for understanding the rhetorical nature of narrative information visualizations are presented, informed by the rigorous application of concepts from critical theory, semiotics, journalism, and political theory. We draw attention to how design tactics represent additions or omissions of information at various levels-the data, visual representation, textual annotations, and interactivity-and how visualizations denote and connote phenomena with reference to unstated viewing conventions and codes. Classes of rhetorical techniques identified via a systematic analysis of recent narrative visualizations are presented, and characterized according to their rhetorical contribution to the visualization. We describe how designers and researchers can benefit from the potentially positive aspects of visualization rhetoric in designing engaging, layered narrative visualizations and how our framework can shed light on how a visualization design prioritizes specific interpretations. We identify areas where future inquiry into visualization rhetoric can improve understanding of visualization interpretation.

  11. [The tangential segregation of simple and complex cells in the visual cortex and their connections. The universal neocortex modulus].

    Science.gov (United States)

    Chebkasov, S A

    1998-01-01

    Guinea-pig studies testify that in rodents (like in higher mammals) the primary afferents from the thalamus and inferior cortical afferents converge on discrete columns about 200 mcm in size with zones of secondary convergence between them. The cortical columns seem to be primary basic and universal cortical modules, since they have similar dimensions and uniform organization in different mammals. The columns concentrate simple cells (with complex ones among them) and afferent inhibitory neurons; these models are involved in the first stage of cortical integration. Local connections of the primary modules differ from those of the secondary intermediary zones, which are poor narrow in rodents. The intermediary zones progressively develop in phylogeny, and in higher mammals they excel the primary modules in dimensions.

  12. Research progress in visualizing single molecule in live cells%活细胞内单分子视踪检测研究进展

    Institute of Scientific and Technical Information of China (English)

    孙德平; 杨凯

    2008-01-01

    对细胞生物学与分子生物学中有关分子事件和相互作用的认识,大部分都是在非活体条件下、忽略分子事件全过程所得到的研究结果,并且这些研究结果是基于被研究的所有单分子在相同的环境以相同的方式运动这个不真实的假设.随着量子点(QDs)等新的荧光物质和抗体模拟物等小分子抗体连接后制备成的新型探针的出现,以及转导外源性荧光探针进入活细胞内的各种方法的发展,再加上优良的荧光成像系统和快速灵敏的荧光采集系统在生命科学中的应用,使得视踪活细胞单个生物分子的轨迹、运动及多种单分子的相互作用成为现实.单分子水平的视踪研究为细胞生物学和分子生物学的研究打开了新的篇章.概述了目前活细胞内单分子视踪技术的研究进展并评价了其发展前景.%Our knowledge of molecular events and interactions in cell biology and molecular biology are mostly based on findings in nonliving body condition while ignoring the whole process of the events,which inevitably involves false assumption that all molecules behave in the same environment and in the same way. With the emergence of new fluorescent molecular probes prepared through linking new fluorescent material such as quantum dots (QDs) to micromolecule antibody such as antibody simulacrum, the development of methods for transducing extrinsic fluorescent probes into live cells,the application of laser technology, the finer fluorescent micro-imaging system, and the fast and sensitive fluorescence collection system in the application of life sciences, it is now possible to image the trajectory, kinetic behavior, and interaction of biomolecules in live cells. Visualizing single molecule in live cells has opened a new chapter for cell biology and molecular biology. Recent research developments and foreground of single molecule visualizing are discussed in this article.

  13. Fluorescence molecular tomography enables in vivo visualization and quantification of nonunion fracture repair induced by genetically engineered mesenchymal stem cells.

    Science.gov (United States)

    Zilberman, Yoram; Kallai, Ilan; Gafni, Yossi; Pelled, Gadi; Kossodo, Sylvie; Yared, Wael; Gazit, Dan

    2008-04-01

    Fluorescence molecular tomography (FMT) is a novel tomographic near-infrared (NIR) imaging modality that enables 3D quantitative determination of fluorochrome distribution in tissues of live small animals at any depth. This study demonstrates a noninvasive, quantitative method of monitoring engineered bone remodeling via FMT. Murine mesenchymal stem cells overexpressing the osteogenic gene BMP2 (mMSCs-BMP2) were implanted into the thigh muscle and into a radial nonunion bone defect model in C3H/HeN mice. Real-time imaging of bone formation was performed following systemic administration of the fluorescent bisphosphonate imaging agent OsteoSense, an hydroxyapatite-directed bone-imaging probe. The mice underwent imaging on days 7, 14, and 21 postimplantation. New bone formation at the implantation sites was quantified using micro-computed tomography (micro-CT) imaging. A higher fluorescent signal occurred at the site of the mMSC-BMP2 implants than that found in controls. Micro-CT imaging revealed a mass of mature bone formed in the implantation sites on day 21, a finding also confirmed by histology. These findings highlight the effectiveness of FMT as a functional platform for molecular imaging in the field of bone regeneration and tissue engineering.

  14. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    Science.gov (United States)

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.

  15. Why Teach Visual Culture?

    Science.gov (United States)

    Passmore, Kaye

    2007-01-01

    Visual culture is a hot topic in art education right now as some teachers are dedicated to teaching it and others are adamant that it has no place in a traditional art class. Visual culture, the author asserts, can include just about anything that is visually represented. Although people often think of visual culture as contemporary visuals such…

  16. Visualizing Gene Expression In Situ

    Energy Technology Data Exchange (ETDEWEB)

    Burlage, R.S.

    1998-11-02

    Visualizing bacterial cells and describing their responses to the environment are difficult tasks. Their small size is the chief reason for the difficulty, which means that we must often use many millions of cells in a sample in order to determine what the average response of the bacteria is. However, an average response can sometimes mask important events in bacterial physiology, which means that our understanding of these organisms will suffer. We have used a variety of instruments to visualize bacterial cells, all of which tell us something different about the sample. We use a fluorescence activated cell sorter to sort cells based on the fluorescence provided by bioreporter genes, and these can be used to select for particular genetic mutations. Cells can be visualized by epifluorescent microscopy, and sensitive photodetectors can be added that allow us to find a single bacterial cell that is fluorescent or bioluminescent. We have also used standard photomultipliers to examine cell aggregates as field bioreporter microorganisms. Examples of each of these instruments show how our understanding of bacterial physiology has changed with the technology.

  17. Mechanisms of early visual processing in the medulla of the locust optic lobe: how self-inhibition, spatial-pooling, and signal rectification contribute to the properties of transient cells.

    Science.gov (United States)

    Osorio, D

    1991-10-01

    In the arthropod medulla, which is the second ganglion on the afferent visual pathway, a column of about 40 cells represents each point in space (i.e. compound eye facet). Some stages of visual processing underlying the responses of one class of cells in the locust medulla have been identified. These transient cells give very similar responses to intensity increments and decrements, and also to pulses and steps; there is no spontaneous activity and a stimulus causes one or two spikes to fire at fixed latencies. Movement, however, produces a prolonged spike discharge by successive excitation of subunits within the receptive field. One of the main features of the transient cells' responses is a self-inhibition which attenuates responses to successive stimuli at one point. This inhibition is restricted to the outputs of single receptor (rhabdom), it decays after about 100 ms, and is polarity sensitive so that stimuli of one polarity (e.g. dimming) do not inhibit responses to stimuli of the opposite polarity (e.g. brightening). The inhibition effectively alters the contrast threshold of the cells, because after adaptation with stimuli of one contrast, a modest (less than 20%) increase in contrast is sufficient to elicit an unadapted response. Transient cells are not directionally selective and there are no local spatio-temporal interactions of the kind necessary for directional selectivity. But, by analogy with the directional veto in directionally selective cells in the rabbit retina (Barlow & Levick, 1965), self-inhibition is suggested as a mechanism of non-directional motion detection. After the inhibition, there is some spatial pooling of signals which is followed by rectification. The transient cells' spiking outputs could abstract a refined subset of visual information which may encode the presence, but not the direction, amplitude, or polarity of moving object borders.

  18. Acyl-CoA binding protein expression is fiber type- specific and elevated in muscles from the obese insulin-resistant Zucker rat.

    Science.gov (United States)

    Franch, Jesper; Knudsen, Jens; Ellis, Bronwyn A; Pedersen, Preben K; Cooney, Gregory J; Jensen, Jørgen

    2002-02-01

    Accumulation of acyl-CoA is hypothesized to be involved in development of insulin resistance. Acyl-CoA binds to acyl-CoA binding protein (ACBP) with high affinity, and therefore knowledge about ACBP concentration is important for interpreting acyl-CoA data. In the present study, we used a sandwich enzyme-linked immunosorbent assay to quantify ACBP concentration in different muscle fiber types. Furthermore, ACBP concentration was compared in muscles from lean and obese Zucker rats. Expression of ACBP was highest in the slow-twitch oxidative soleus muscle and lowest in the fast-twitch glycolytic white gastrocnemius (0.46 +/- 0.02 and 0.16 +/- 0.005 microg/mg protein, respectively). Expression of ACBP was soleus > red gastrocnemius > extensor digitorum longus > white gastrocnemius. Similar fiber type differences were found for carnitine palmitoyl transferase (CPT)-1, and a correlation was observed between ACBP and CPT-1. Muscles from obese Zucker rats had twice the triglyceride content, had approximately twice the long-chain acyl CoA content, and were severely insulin resistant. ACBP concentration was approximately 30% higher in all muscles from obese rats. Activities of CPT-1 and 3-hydroxy-acyl-CoA dehydrogenase were increased in muscles from obese rats, whereas citrate synthase activity was similar. In conclusion, ACBP expression is fiber type-specific with the highest concentration in oxidative muscles and the lowest in glycolytic muscles. The 90% increase in the concentration of acyl-CoA in obese Zucker muscle compared with only a 30% increase in the concentration of ACBP supports the hypothesis that an increased concentration of free acyl-CoA is involved in the development of insulin resistance.

  19. Human papillomavirus infections in Mexican women with normal cytology, precancerous lesions, and cervical cancer: type-specific prevalence and HPV coinfections.

    Science.gov (United States)

    Aguilar-Lemarroy, Adriana; Vallejo-Ruiz, Verónica; Cortés-Gutiérrez, Elva I; Salgado-Bernabé, Manuel Eduardo; Ramos-González, Norma Patricia; Ortega-Cervantes, Laura; Arias-Flores, Rafael; Medina-Díaz, Irma M; Hernández-Garza, Fernando; Santos-López, Gerardo; Piña-Sánchez, Patricia

    2015-05-01

    The prevalence and genotype distribution of human papillomavirus (HPV) provides the basis for designing HPV prevention programs. The prevalence rates of type-specific HPV and coinfections in samples of Mexican women were investigated in 822 women aged 18-87 years. HPV detection was performed using a Linear Array™ genotyping test. HPV infection was found in 12.4% of controls, 46.3% of those with cervical intraepithelial neoplasia 1, and 100% of those with cervical intraepithelial neoplasia 3 or cervical cancer. HPV 16 was the most prevalent type in all diagnosis groups. The HPV types most frequently found in cervical cancers were 16, 18, 45, 52, 58, and 39; HPV types 16, 62, 51, 84, 18, 53, and CP6108 were the most prevalent in control women. Considering HPV-positive samples only, coinfections occurred most often in controls (63%) and were less frequent in those with cervical cancer (26%). The most frequent viral types in coinfections with HPV 16 in control women were HPV 62, 51, and 84; in women with cervical cancers, HPV 18, 39, and 70 were most common. In conclusion, in addition to HPV types 16 and 18, types 45, 39, 58, 52, and 71 were found in cervical cancers in Mexican women (78%); among them, only 65% were attributable to HPV types 16 and 18. Therefore, it is necessary to consider these viral types in the design of new vaccines, and to determine whether certain HPV types coinfecting with HPV 16 in precursor lesions determine tumor progression or regression.

  20. Ovalbumin expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type-specific responses.

    Science.gov (United States)

    Zhao, J P; Zhang, Q; Jiao, H C; Wang, X J; Jiang, M J; Luo, H; Lin, H

    2016-10-01

    Three trials were performed to evaluate the association of ovalbumin (OVA) abundance in the oviduct magnum with egg production and the underlying regulatory mechanism by glucocorticoids. In trial 1, twenty Hy-Line Brown layers (56-60 weeks of age) with different combinations (n = 5/combination) of laying rate (high or low) and egg weight (high or low) were selected from an initial group of 300. An upregulated expression of magnum OVA was observed (p hens with higher laying rate, regardless of egg weight. In trial 2, eighty Hy-Line Brown layers (80-90 weeks of age) were subjected to the forced moulting (n = 8). The abundance of OVA transcript and protein in the magnum was significantly decreased during moulting (p old Hy-Line Brown layers were kept individually (n = 15) in the following conditions for 10 days: constant optimal ambient temperature at 23 °C and ad libitum feeding, high ambient temperature at 32 °C for 6 h/day (10:00-16:00) and ad libitum feeding (32AL), and constant optimal ambient temperature at 23 °C and pair-fed to the 32AL hens. In spite of elevated corticosterone in circulation, OVA synthesis, blood oestrogen and laying rate were not affected by heat exposure (p > 0.05). These results allow concluding that OVA expression in the oviduct magnum of hens is related to the rate of egg laying and shows distinct stress-type-specific responses.

  1. The I4895T mutation in the type 1 ryanodine receptor induces fiber-type specific alterations in skeletal muscle that mimic premature aging.

    Science.gov (United States)

    Boncompagni, Simona; Loy, Ryan E; Dirksen, Robert T; Franzini-Armstrong, Clara

    2010-12-01

    The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized age-dependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca(2+) release in heterozygous Ryr1(I4895T/WT) (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca(2+) release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber-type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.

  2. Investigating visual analogies for visual insight problems

    OpenAIRE

    Corina Sas; Eric Luchian; Linden Ball

    2010-01-01

    Much research has focused on the impact of analogies in insight problem solving, but less work has investigated how the visual analogies for insight are actually constructed. Thus, it appears that in the search for their facilitative impact on the incubation effect, the understanding of what makes good visual analogies has somehow been lost. This paper presents preliminary work of constructing a set of 6 visual analogies and evaluating their impact on solving the visual problem of eight coins...

  3. Data Visualization and Infographics

    OpenAIRE

    Prepared by Mathematica Policy Research

    2014-01-01

    Data visualization translates complex ideas and concepts into a simple visual context. Patterns, trends, and relationships that might go undetected in text are conveyed at a glance in effective data visualization.

  4. Cortical Visual Impairment

    Science.gov (United States)

    ... Frequently Asked Questions Español Condiciones Chinese Conditions Cortical Visual Impairment En Español Read in Chinese What is cortical visual impairment? Cortical visual impairment (CVI) is a decreased ...

  5. Altered anterior visual system development following early monocular enucleation

    Directory of Open Access Journals (Sweden)

    Krista R. Kelly

    2014-01-01

    Conclusions: The novel finding of an asymmetry in morphology of the anterior visual system following long-term survival from early monocular enucleation indicates altered postnatal visual development. Possible mechanisms behind this altered development include recruitment of deafferented cells by crossing nasal fibres and/or geniculate cell retention via feedback from primary visual cortex. These data highlight the importance of balanced binocular input during postnatal maturation for typical anterior visual system morphology.

  6. A visual approach to proteomics.

    Science.gov (United States)

    Nickell, Stephan; Kofler, Christine; Leis, Andrew P; Baumeister, Wolfgang

    2006-03-01

    Cryo-electron tomography is an emerging imaging technique that has unique potential for molecular cell biology. At the present resolution of 4-5 nm, large supramolecular structures can be studied in unperturbed cellular environments and, in the future, it will become possible to map molecular landscapes inside cells in a more comprehensive manner. 'Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions.

  7. Snowflake Visualization

    Science.gov (United States)

    Bliven, L. F.; Kucera, P. A.; Rodriguez, P.

    2010-12-01

    NASA Snowflake Video Imagers (SVIs) enable snowflake visualization at diverse field sites. The natural variability of frozen precipitation is a complicating factor for remote sensing retrievals in high latitude regions. Particle classification is important for understanding snow/ice physics, remote sensing polarimetry, bulk radiative properties, surface emissivity, and ultimately, precipitation rates and accumulations. Yet intermittent storms, low temperatures, high winds, remote locations and complex terrain can impede us from observing falling snow in situ. SVI hardware and software have some special features. The standard camera and optics yield 8-bit gray-scale images with resolution of 0.05 x 0.1 mm, at 60 frames per second. Gray-scale images are highly desirable because they display contrast that aids particle classification. Black and white (1-bit) systems display no contrast, so there is less information to recognize particle types, which is particularly burdensome for aggregates. Data are analyzed at one-minute intervals using NASA's Precipitation Link Software that produces (a) Particle Catalogs and (b) Particle Size Distributions (PSDs). SVIs can operate nearly continuously for long periods (e.g., an entire winter season), so natural variability can be documented. Let’s summarize results from field studies this past winter and review some recent SVI enhancements. During the winter of 2009-2010, SVIs were deployed at two sites. One SVI supported weather observations during the 2010 Winter Olympics and Paralympics. It was located close to the summit (Roundhouse) of Whistler Mountain, near the town of Whistler, British Columbia, Canada. In addition, two SVIs were located at the King City Weather Radar Station (WKR) near Toronto, Ontario, Canada. Access was prohibited to the SVI on Whistler Mountain during the Olympics due to security concerns. So to meet the schedule for daily data products, we operated the SVI by remote control. We also upgraded the

  8. Visuals for Information.

    Science.gov (United States)

    Pettersson, Rune

    This report focuses on the visual component of verbo-visual literacy, a communications concept involving the production, transmission, and perception of verbal and visual images. Five current problem areas in verbal-visual research are introduced and discussed: (1) communication (communication models, media consumption, new media, the information…

  9. Using transparency in visualization

    OpenAIRE

    2011-01-01

    Over the last two decades, there have been a growing number of applications for transparency in visualization. Transparency is a visual feature that provides solutions to certain fundamental visualization problems. Currently, there is insufficient research regarding the benefits and the limitations of