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Sample records for single mesophyll cells

  1. Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate

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    Misra, Biswapriya B.; de Armas, Evaldo; Tong, Zhaohui; Chen, Sixue

    2015-01-01

    Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3 -). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3 - responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3 -. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage. PMID:26641455

  2. Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

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    van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

    2015-04-01

    Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. FUNCTION OF MALATDEHYDROGENASE COMPLEX OF MAIZE MESOPHYLL AND BUNDLE SHEATH CELLS UNDER SALT STRESS CONDITION

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    Еprintsev А.Т.

    2006-12-01

    Full Text Available Salt-induced changes in malatdehydrogenase system activity make the essential contribution to cell adaptation to stress condition. The enzyme systems of C4-plants are most interesting due to their ability for adaptation to environment conditions. The role of separate components of malatdehydrogenase complex of mesophyll and bundle sheath cells of corn in formation of adaptive reaction in stressful conditions is investigated in presented work.The activation of all enzymes of malatdehydrogenase system and the subsequent decrease in their activity was observed in mesophyll durring the first stage of adaptation to salt influence. In bundle sheath cells such parameters are differed from control less essentially. Fast accumulation of piruvate in cells and malate in both investigated tissues was induced. The further salinity led to falling of concentration this intermediate. The concentration of piruvate was below control level, and it was raised by the end of an exposition.The results show that sodium chloride causes induction of Krebs-cycle in mesophyll and bundle sheath cells of corn and intensification of Hatch-Slack cycle. The described differences in function malatdehydrogenase systems of mesophyll and bundle sheath cells of leaves of corn under salinity mainly consist of the activity of enzymes of a studied complex in bundle sheath cells is subject to the minimal changes in comparison with mesophyll. Role of this enzymesystem in mechanisms of adaptive reaction of various tissues of corn to salt stress is discussed.

  4. Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM.

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    Oi, Takao; Enomoto, Sakiko; Nakao, Tomoyo; Arai, Shigeo; Yamane, Koji; Taniguchi, Mitsutaka

    2017-07-01

    Ultrathin sections of rice leaf blades observed two-dimensionally using a transmission electron microscope (TEM) show that the chlorenchyma is composed of lobed mesophyll cells, with intricate cell boundaries, and lined with chloroplasts. The lobed cell shape and chloroplast positioning are believed to enhance the area available for the gas exchange surface for photosynthesis in rice leaves. However, a cell image revealing the three-dimensional (3-D) ultrastructure of rice mesophyll cells has not been visualized. In this study, a whole rice mesophyll cell was observed using a focused ion beam scanning electron microscope (FIB-SEM), which provides many serial sections automatically, rapidly and correctly, thereby enabling 3-D cell structure reconstruction. Rice leaf blades were fixed chemically using the method for conventional TEM observation, embedded in resin and subsequently set in the FIB-SEM chamber. Specimen blocks were sectioned transversely using the FIB, and block-face images were captured using the SEM. The sectioning and imaging were repeated overnight for 200-500 slices (each 50 nm thick). The resultant large-volume image stacks ( x = 25 μm, y = 25 μm, z = 10-25 μm) contained one or two whole mesophyll cells. The 3-D models of whole mesophyll cells were reconstructed using image processing software. The reconstructed cell models were discoid shaped with several lobes around the cell periphery. The cell shape increased the surface area, and the ratio of surface area to volume was twice that of a cylinder having the same volume. The chloroplasts occupied half the cell volume and spread as sheets along the cell lobes, covering most of the inner cell surface, with adjacent chloroplasts in close contact with each other. Cellular and sub-cellular ultrastructures of a whole mesophyll cell in a rice leaf blade are demonstrated three-dimensionally using a FIB-SEM. The 3-D models and numerical information support the hypothesis that rice mesophyll

  5. Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

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    Elzenga, J.T.M.; van Volkenburgh, E.

    Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The

  6. Characterization of a light-controlled anion channel in the plasma membrane of mesophyll cells of pea

    NARCIS (Netherlands)

    Elzenga, J.T.M.; Volkenburgh Van, E

    In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp

  7. Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass

    Science.gov (United States)

    2014-01-01

    Background Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. Results The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. Conclusions In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants. PMID:25209261

  8. Physiological implications of seasonal variation in membrane-associated calcium in red spruce mesophyll cells

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    D.H. DeHayes; P.G. Schaberg; G.J. Hawley; C.H. Borer; J.R. Cumming; J.R. Strimbeck

    1997-01-01

    We examined the pattern of seasonal variation in total foliar calcium (Ca) pools and plasma membrane-associated Ca (mCa) in mesophyll cells of current-year and 1-year-old needles of red spruce (Picea rubens Sarg.) and the relationship between mCa and total foliar Ca on an individual plant and seasonal basis. Foliar samples were collected from...

  9. Effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of summer maize.

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    Ren, Baizhao; Cui, Haiyan; Camberato, James J; Dong, Shuting; Liu, Peng; Zhao, Bin; Zhang, Jiwang

    2016-08-01

    A field experiment was conducted to study the effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of two summer maize hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The ambient sunlight treatment was used as control (CK) and shading treatments (40 % of ambient sunlight) were applied at different growth stages from silking (R1) to physiological maturity (R6) (S1), from the sixth leaf stage (V6) to R1 (S2), and from seeding to R6 (S3), respectively. The net photosynthetic rate (P n) was significantly decreased after shading. The greatest reduction of P n was found at S3 treatment, followed by S1 and S2 treatments. P n of S3 was decreased by 59 and 48 % for DH605, and 39 and 43 % for ZD958 at tasseling and milk-ripe stages, respectively, compared to that of CK. Additionally, leaf area index (LAI) and chlorophyll content decreased after shading. In terms of mesophyll cell ultrastructure, chloroplast configuration of mesophyll cells dispersed, and part of chloroplast swelled and became circular. Meanwhile, the major characteristics of chloroplasts showed poorly developed thylakoid structure at the early growth stage, blurry lamellar structure, loose grana, and a large gap between slices and warping granum. Then, plasmolysis occurred in mesophyll cells and the endomembrane system was destroyed, which resulted in the dissolution of cell membrane, karyotheca, mitochondria, and some membrane structures. The damaged mesophyll cell ultrastructure led to the decrease of photosynthetic capacity, and thus resulted in significant yield reduction by 45, 11, and 84 % in S1, S2, and S3 treatments, respectively, compared to that of CK.

  10. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

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    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  11. Signal function of cytokinin 6-benzylaminopurine in the reaction of Triticum aestivum L. mesophyll cells to hyperthermia

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    M. M. Musienko

    2014-12-01

    Full Text Available The signaling effect of 6-benzylaminopurine (BAP on leaf mesophyll cells of Triticum aestivum L. under hyperthermic conditions was studied­. It was found that BAP regulated photosynthetic pigment, hydrogen peroxide content and activity of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase and catalase under high-temperature conditions. The additive effect of BAP and high temperature on the activation of cell antioxidant systems was demonstrated. BAP regulated reducing processes in mesophyll leaf cells under high-temperature conditions.

  12. Uptake of /sup 86/Rb/sup +/ into photoautotrophic mesophyll cells of Papaver somniferum

    Energy Technology Data Exchange (ETDEWEB)

    Kaiser, W.M.; Jeschke, W.D.; Hartung, W.

    1982-06-01

    Uptake of /sup 86/Rb/sup +/, used as a tracer for potassium, into isolated photoautotrophic mesophyll cells of Papaver somniferum was weakly but consistently stimulated in the light. It showed mono-phasic saturation kinetics with a pH optimum of 7.0, a Vsub(max) of 6.7 ..mu..mol mg/sup -1/ Chl x h/sup -1/ and a Ksub(m) of 2.7 mmol l/sup -1/. Different anions as Cl/sup -/, NO/sub 3//sup -/ and PO/sub 4//sup 3 -/ had no effects on /sup 86/Rb/sup +/ uptake. Sodium ions influenced Rb/sup +/-uptake very weakly, indicating a high K/sup +/ -specificity of the mesophyll cell plasmalemma. Fusicoccin stimulated /sup 86/Rb/sup +/ -uptake strongly whereas abscisic acid inhibited uptake only following preincubation for two hours. Nitrite, CCCP and Dio-9 inhibited /sup 86/Rb/sup +/-uptake which gives evidence that this process is dependent on intact pH-gradients within the cells and on ATP-formation.

  13. Evidence for a specific glutamate/H+ cotransport in isolated mesophyll cells

    International Nuclear Information System (INIS)

    McCutcheon, S.L.; Bown, A.W.

    1987-01-01

    Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO 4 . Immediate alkalinization of the medium occurred on the addition of 1 millimolar concentrations of L-glutamate (Glu) and its analog L-methionine-D,L-sulfoximine (L-MSO). D-Glu and the L isomers of the protein amino acids did not elicit alkalinization. L-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar L-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H + /10 6 cells minute. L-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of L-[U- 14 C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by L-MSO. L-Glu had no influence on K + efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific L-Glu/proton uptake process is present in Asparagus mesophyll cells

  14. Air pollution effects on the ultrastructure of Phlomis fruticosa mesophyll cells

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    Psaras, G.K.; Christodoulakis, N.S.

    1987-04-01

    Plant physiologists and environmental scientists suggest that a basic effect of air pollution on plants leads towards the minimization of their productivity. On the other hand the action of individual pollutants on intact plants has been studied from biochemical as well as structural viewpoint. Thus the study of plant responses to SO/sub 2/ exposure revealed that this agent causes acute and chronic injury. Chronic injury results in chlorosis and subsequent necrosis due to destruction of chlorophylls and final chloroplast lysis. It has been documented that ultrastructural characteristics of leaves are affected prior to any visible injury. Electron microscope examination of SO/sub 2/ fumigated plant-attached leaves of Vicia faba revealed chloroplast thylakoids starting to swell whilst photosynthesis rate was drastically reduced. The first light microscope-detected effects of air pollution on the leaf structure of plants common in natural ecosystems of Athens metropolitan area, have been reported. A chlorosis phenomenon in Urginea maritima leaves as well as an indication of detrimental effects of Phlomis fruticosa mesophyll chloroplasts were documented. In this work further investigation has been undertaken in order to elucidate the precise effects of air pollution on the ultrastructure of the photosynthesizing mesophyll cells.

  15. Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells.

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    Thayer, S S; Huffaker, R C

    1984-05-01

    The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.

  16. Effects of water stress on photosynthetic electron transport, photophosphorylation, and metabolite levels of Xanthium strumarium mesophyll cells.

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    Sharkey, T D; Badger, M R

    1982-12-01

    Several component processes of photosynthesis were measured in osmotically stressed mesophyll cells of Xanthium strumarium L. The ribulose-1,5-bisphosphate regeneration capacity was reduced by water stress. Photophoshorylation was sensitive to water stress but photosynthetic electron transport was unaffected by water potentials down to-40 bar (-4 MPa). The concentrations of several intermediates of the photosynthetic carbon-reduction cycle remained relatively constant and did not indicate that ATP supply was limiting photosynthesis in the water-stressed cells.

  17. Ultrastructural response of cabbage outer leaf mesophyll cells (Brassica oleracea L. to excess of nickel

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    Jolanta Molas

    2014-01-01

    Full Text Available Changes in the structure and in the ultrastructure of cabbage outer leaf mesophyll cells [Brassica oleracea L.] cv. Sława from Enkhouizen were examined by means of light and electron microscopy. The examined plants were grown on the basic Murashige and Skoog medium with addition of excesive concentrations of nickel (added as NiSO4 x 7H2O,i.e. Ni 5, Ni 10 and Ni 20 mg/dm3. In Ni 5 mg samples mainly adaptation changes to the conditions of stress were observed. These changes were manifested by the increase of cytoplasm content and by cytoplasm vacuolization, by the increase of nucleus and nucleous volume, nucleolus vacuolization, the increase of plasmalemma invaginations and of the amount of rough ER, by the central arrangement of smooth ER and of the thylakoids of chloroplasts; it was also shown by the growth of the number of mitochondria and of peroxisomes in the cell. In Ni 10 mg samples, apart from adaptation changes, such as the increase of the nucleus volume, increase of plasmalemma invaginations, cytoplasm and nucleolus vacuolization, degeneration changes were also observed. They concerned mainly the nucleus (the increasing amount of condensed chromatin, ER (swelling and fragmentation of rER and sER, mitochondrium (swelling and reduction of cristae, Golgi apparatus (disintegration and decay and chloroplasts (changes of shape, swelling and reduction of thylakoids, disappearance of starch and presence of big plastoglobuli. In Ni 20 mg samples cell protoplasts were in different stages of degeneration and the cell organelles that were identifiable, were usually damaged.

  18. Inorganic carbon uptake during photosynthesis. II. Uptake by isolated Asparagus mesophyll cells during isotopic disequilibrium

    International Nuclear Information System (INIS)

    Espie, G.S.; Owttrim, G.W.; Colman, B.

    1986-01-01

    The species of inorganic carbon (CO 2 or HCO 3 - ) taken up as a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO 2 or HCO 3 - transport, during steady state photosynthesis, is achieved by monitoring the changes (by 14 C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO 2 or HCO 3 - transport) and experimental time-courses of 14 C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO 2 , rather than HCO 3 - , is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO 2 is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO 3 - transport, as the incorporation of 14 C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO 2 uptake alone. The contribution of HCO 3 - to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO 3 - concentration. The evidence for direct HCO 3 - transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of 14 C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO 2 , which is partially alleviated by a high extracellular concentration of HCO 3 -

  19. Stomatal responses to flooding of the intercellular air spaces suggest a vapor-phase signal between the mesophyll and the guard cells.

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    Sibbernsen, Erik; Mott, Keith A

    2010-07-01

    Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO(2). These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K(+) in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.

  20. Stomatal Responses to Flooding of the Intercellular Air Spaces Suggest a Vapor-Phase Signal Between the Mesophyll and the Guard Cells1[OA

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    Sibbernsen, Erik; Mott, Keith A.

    2010-01-01

    Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO2. These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K+ in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light. PMID:20472750

  1. Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.

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    Majeran, Wojciech; Zybailov, Boris; Ytterberg, A Jimmy; Dunsmore, Jason; Sun, Qi; van Wijk, Klaas J

    2008-09-01

    Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are

  2. Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays.

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    Giannoutsou, E; Sotiriou, P; Apostolakos, P; Galatis, B

    2013-10-01

    The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs. Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy. Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes. The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule

  3. A new mechanism for the regulation of stomatal aperture size in intact leaves: accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba

    International Nuclear Information System (INIS)

    Lu, P.; Outlaw, W.H. Jr.; Smith, B.G.; Freed, G.A.

    1997-01-01

    At various times after pulse-labeling broad bean (Vicia faba L.) leaflets with 14CO2, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents, whereas those from rinsed peels contained only symplastic contents. Sucrose (Suc)-specific radioactivity peaked (111 GBq mol-1) in palisade cells at 20 min. In contrast, the 14C content and Suc-specific radioactivity were very low in guard cells for 20 min, implying little CO2 incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum Suc-specific radioactivity (204 GBq mol-1) and a high Suc influx rate (0.05 pmol stoma-1 min-1). These and other comparisons implied the presence of (a) multiple Suc pools in mesophyll cells, (b) a localized mesophyll-apoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived Suc in guard-cell walls sufficient to diminish stomatal opening by approximately 3 micrometers. Factors expected to enhance Suc accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic Suc concentration, which is elevated when mesophyll Suc efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal aperture size by this previously unrecognized mechanism

  4. Intracellular position of mitochondria and chloroplasts in bundle sheath and mesophyll cells of C3 grasses in relation to photorespiratory CO2 loss

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    Yuto Hatakeyama

    2016-10-01

    Full Text Available In C3 plants, photosynthetic efficiency is reduced by photorespiration. A part of CO2 fixed during photosynthesis in chloroplasts is lost from mitochondria during photorespiration by decarboxylation of glycine by glycine decarboxylase (GDC. Thus, the intracellular position of mitochondria in photosynthetic cells is critical to the rate of photorespiratory CO2 loss. We investigated the intracellular position of mitochondria in parenchyma sheath (PS and mesophyll cells of 10 C3 grasses from 3 subfamilies (Ehrhartoideae, Panicoideae, and Pooideae by immunostaining for GDC and light and electron microscopic observation. Immunostaining suggested that many mitochondria were located in the inner half of PS cells and on the vacuole side of chloroplasts in mesophyll cells. Organelle quantification showed that 62–75% of PS mitochondria were located in the inner half of cells, and 62–78% of PS chloroplasts were in the outer half. In mesophyll cells, 61–92% of mitochondria were positioned on the vacuole side of chloroplasts and stromules. In PS cells, such location would reduce the loss of photorespiratory CO2 by lengthening the path of CO2 diffusion and allow more efficient fixation of CO2 from intercellular spaces. In mesophyll cells, it would facilitate scavenging by chloroplasts of photorespiratory CO2 released from mitochondria. Our data suggest that the PS cells of C3 grasses have already acquired an initial structure leading to proto-Kranz and further C3–C4 intermediate anatomy. We also found that in the Pooideae, organelle positioning in PS cells on the phloem side resembles that in mesophyll cells.

  5. Cyst(e)ine Is the Transport Metabolite of Assimilated Sulfur from Bundle-Sheath to Mesophyll Cells in Maize Leaves1

    Science.gov (United States)

    Burgener, Marta; Suter, Marianne; Jones, Stephanie; Brunold, Christian

    1998-01-01

    The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, γ-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types. PMID:9536048

  6. Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

    Science.gov (United States)

    Sotiriou, P; Giannoutsou, E; Panteris, E; Apostolakos, P; Galatis, B

    2016-03-01

    This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril

  7. Early H2O2 Accumulation in Mesophyll Cells Leads to Induction of Glutathione during the Hyper-Sensitive Response in the Barley-Powdery Mildew Interaction1

    Science.gov (United States)

    Vanacker, Helene; Carver, Tim L.W.; Foyer, Christine H.

    2000-01-01

    H2O2 production and changes in glutathione, catalase, and peroxidase were followed in whole-leaf extracts from the susceptible (AlgS [Algerian/4* (F14) Man.(S)]; ml-a1 allele) and resistant (AlgR [Algerian/4* (F14) Man.(R)]; Ml-a1 allele) barley (Hordeum vulgare) isolines between 12 and 24 h after inoculation with powdery mildew (Blumeria graminis [DC]. Speer [syn. Erysiphe graminis DC] f.sp hordei Marchal). Localized papilla responses and cell death hypersensitive responses were not observed within the same cell. In hypersensitive response sites, H2O2 accumulation first occurred in the mesophyll underlying the attacked epidermal cell. Subsequently, H2O2 disappeared from the mesophyll and accumulated around attacked epidermal cells. In AlgR, transient glutathione oxidation coincided with H2O2 accumulation in the mesophyll. Subsequently, total foliar glutathione and catalase activities transiently increased in AlgR. These changes, absent from AlgS, preceded inoculation-dependent increases in peroxidase activity that were observed in both AlgR and AlgS at 18 h. An early intercellular signal precedes H2O2, and this elicits anti-oxidant responses in leaves prior to events leading to death of attacked cells. PMID:10938348

  8. Effects of herbicides on /sup 14/CO/sub 2/ fixation in isolated mesophyll cells from Beta vulgaris (sugar beet) and Chenopodium album

    Energy Technology Data Exchange (ETDEWEB)

    Baumann, G; Guenther, G [Paedagogische Hochschule Karl Liebknecht, Potsdam (German Democratic Republic)

    1979-01-01

    10/sup -4/ - 10/sup -6/ molar solutions of herbicides (atrazine, 2,4-D, desmetryne, diallate, diquat, feuron, lenacil, NaTa, paraquat, phenmedipham, prometryne, propham, pyrazone, and simazine) cause similar inhibitory effects on the photosynthetic /sup 14/CO/sub 2/ fixation in isolated mesophyll cells from Chenopodium album and Beta vulgaris. Correlatdion between inhibition and herbicide resistance of the whole plants could be realized for lenacil only.

  9. Identification of the TaBTF3 gene in wheat (Triticum aestivum L.) and the effect of its silencing on wheat chloroplast, mitochondria and mesophyll cell development.

    Science.gov (United States)

    Ma, Hong-Zhen; Liu, Guo-Qin; Li, Cheng-Wei; Kang, Guo-Zhang; Guo, Tian-Cai

    2012-10-05

    The full-length cDNA (882bp) and DNA (1742bp) sequences encoding a basic transcription factor 3, designated as TaBTF3, were first isolated from common wheat (Triticum aestivum L.). Subcellular localization studies revealed that the TaBTF3 protein was mainly located in the cytoplasm and nucleus. In TaBTF3-silenced transgenic wheat seedlings obtained using the Virus-induced gene silencing (VIGS) method, the chlorophyll pigment content was markedly reduced. However, the malonaldehyde (MDA) and H(2)O(2) contents were enhanced, and the structure of the wheat mesophyll cell was seriously damaged. Furthermore, transcripts of the chloroplast- and mitochondrial-encoded genes were significantly reduced in TaBTF3-silenced transgenic wheat plants. These results suggest that the TaBTF3 gene might function in the development of the wheat chloroplast, mitochondria and mesophyll cell. This paper is the first report to describe the involvement of TaBTF3 in maintaining the normal plant mesophyll cell structure. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Intracellular compartimentation of abscisic acid (ABA) in guard cells and mesophyll cells under exposure to SO sub 2. Kompartimentierung von Abscisinsaeure (ABA) in Schliess- und Mesophyllzellen unter SO sub 2 -Belastung

    Energy Technology Data Exchange (ETDEWEB)

    Baier, M.; Daeter, W.; Hartung, W. (Wuerzburg Univ. (Germany, F.R.). Lehrstuhl fuer Botanik 1)

    1989-07-01

    The effect of SO{sub 2} on the intracellular compartimentation of ABA in guard cells and mesophyll cells of Valerianella locusta was investigated, using the efflux compartmental analysis, as described by Behl and Hartung (1986). The cytoplasmic ABA content of the guard cells was reduced drastically by 6 {mu}molxm{sup -3} SO{sub 2} (20% of the controls). The vacuolar content was decreased less dramatically (70% of the controls). The ABA distribution of mesophyll cells remained uneffected by 6 {mu}molxm{sup -3} SO{sub 2}. The SO{sub 2} effects are explained by an acidification of the compartments. (orig.).

  11. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    Directory of Open Access Journals (Sweden)

    Andreia Michelle Smith-Moritz

    2015-08-01

    Full Text Available The CELLULOSE SYNTHASE-LIKE F6 (CslF6 gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG, a cell wall polysaccharide that is hypothesized to be a tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of three day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell was of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  12. Effect of Hf on the fine structure of mesophyll cells from Glycine max, Merr

    Energy Technology Data Exchange (ETDEWEB)

    Wei, L.; Miller, G.W.

    1972-04-01

    A series of ultrastructural changes were observed in soybean leaves fumigated with 40 to 50 ppb of hydrogen fluoride. In the cytoplasm the presence of small vacuoles was the first noticeable initial change. The fragmentation of the vacuolar membrane occurred either simultaneously or followed immediately. Lipid-droplet-like globules and numerous vesicles occurred subsequently in the cytoplasm and increased as the injury became more severe. There was a decrease in polysomes and a detachment of ribosome from the rough endoplasmic reticulum. Free ribosome concentration also decreased as the injury became severe. Mitochondrial modifications involving dilation of outer and cristae membranes followed by reduction of both cristae number and matrix electron density and the disappearance of mitochondrial granules were observed in the chlorotic leaves. Electron dense inclusions accumulated in some mitochondria as well. The first noticeable change observed in the chloroplast was the presence of clusters of phytoferritin granules within the stoma after only 2 days of fumigation. Alterations in nuclear structures were observed in later stages of injury. Numerous small electron dense particles were found on various types of membranes in cells of severely chlorotic leaves. They were distributed on outer mitochondrial membranes, endoplasmic reticula, dictyosomes, tonoplasts, plasmalemma, nuclear envelopes, and disintegrating organelles and vesicles, but were never observed on membranes of chloroplasts and microbodies. The presence of fluoride has attracted the attention of many workers primarily in certain industrial areas where the emitted atmospheric fluoride concentrates and is accumulated by plants initiating injury. 6 references.

  13. Increasing Leaf Vein Density via Mutagenesis in Rice Results in an Enhanced Rate of Photosynthesis, Smaller Cell Sizes and Can Reduce Interveinal Mesophyll Cell Number

    Directory of Open Access Journals (Sweden)

    Aryo B. Feldman

    2017-11-01

    Full Text Available Improvements to leaf photosynthetic rates of crops can be achieved by targeted manipulation of individual component processes, such as the activity and properties of RuBisCO or photoprotection. This study shows that simple forward genetic screens of mutant populations can also be used to rapidly generate photosynthesis variants that are useful for breeding. Increasing leaf vein density (concentration of vascular tissue per unit leaf area has important implications for plant hydraulic properties and assimilate transport. It was an important step to improving photosynthetic rates in the evolution of both C3 and C4 species and is a foundation or prerequisite trait for C4 engineering in crops like rice (Oryza sativa. A previous high throughput screen identified five mutant rice lines (cv. IR64 with increased vein densities and associated narrower leaf widths (Feldman et al., 2014. Here, these high vein density rice variants were analyzed for properties related to photosynthesis. Two lines were identified as having significantly reduced mesophyll to bundle sheath cell number ratios. All five lines had 20% higher light saturated photosynthetic capacity per unit leaf area, higher maximum carboxylation rates, dark respiration rates and electron transport capacities. This was associated with no significant differences in leaf thickness, stomatal conductance or CO2 compensation point between mutants and the wild-type. The enhanced photosynthetic rate in these lines may be a result of increased RuBisCO and electron transport component amount and/or activity and/or enhanced transport of photoassimilates. We conclude that high vein density (associated with altered mesophyll cell length and number is a trait that may confer increased photosynthetic efficiency without increased transpiration.

  14. Effects of potentially acidic air pollutants on the intracellular distribution and transport of plant growth regulators in mesophyll cells of leaves. Consequences on stress- and developmental physiology

    Energy Technology Data Exchange (ETDEWEB)

    Kremer, H.; Pfanz, H.; Hartung, W.

    1987-07-11

    The influence of SO/sub 2/ on the intracellular distribution of abscisic acid (ABA) and indole-acetic acid (IAA) in mesophyll cells of Picea abies, Tsuga americana and Hordeum vulgare was investigated. The compartmentation of ABA and IAA depends on intracellular pH-gradients. The hydrophilic anions ABA and IAA are accumulated in the alkaline cell compartments cytosol and chloroplasts, which act as anion traps for weak acids. Uptake of sulfur dioxide into leaves leads to an acidification of alkaline cell compartments, thus decreasing intracellular pH-gradients. Consequently this results in an increased release of plant growth regulators from the cell interior into the apoplast. Therefore the target cells of plant hormones i.e. meristems and stomates are exposed to altered hormone concentrations. Obviously this influences the regulation of cellular metabolism plant development and growth.

  15. [Effects of light intensities after anthesis on the photosynthetic characteristics and chloroplast ultrastructure in mesophyll cell of summer maize (Zea mays L. )].

    Science.gov (United States)

    Gao, Jia; Cui, Hai Yan; Shi, Jian Guo; Dong, Shu Ting; Liu, Peng; Zhao, Bin; Zhang, Ji Wang

    2018-03-01

    We examined the changes of photosynthetic characteristics and chloroplast ultrastructure in mesophyll cell of summer maize in response to different light intensities in the field, with the summer maize hybrid Denghai 605 as experimental material. Two treatments of both shading (S) and increasing light (L) from flowering to physiological maturity stage were designed, with the ambient sunlight treatment as control (CK). Under shading treatment, poorly developed thylakoid structure, blurry lamellar structure, loose granum, large gap between slices and warping granum were the major characteristics in chloroplast. Meanwhile, photosynthetic rate (P n ), transpiration rate, stomatal conductance, chlorophyll content, and actual photo-chemical efficiency (Φ PSII ) decreased, whereas the maximal photochemical efficiency and non-photochemical quenching increased, which resulted in decreases in grain yield under shading treatment. However, a better development was observed in chloroplasts for L treatment, with the number of grana and lamellae increased and lamellae arranged compactly. In addition, P n and Φ PSII increased under L treatment, which increased grain yield. The chloroplast arrangement dispersed in mesophyll cells and chloroplast ultrastructure was destroyed after shading, and then chlorophyll synthesis per unit leaf area and photosynthetic capacity decreased. In contrast, the number of grana and lamellae increased and lamellae arranged compactly after increasing light, which are beneficial for corn yield.

  16. Influence of pH on the /sup 14/C-labelling pattern after photosynthesis of suspended leaf slices and isolated mesophyll cells from chenopodium album in NaH/sup 14/CO/sub 3/

    Energy Technology Data Exchange (ETDEWEB)

    Baumann, G; Guenther, G [Paedagogische Hochschule Karl Liebknecht, Potsdam (German Democratic Republic). Sektion Chemie/Biologie

    1983-01-01

    Photosynthetic fixation of /sup 14/C from solutions of NaH/sup 14/CO/sub 3/ (at constant concentrations of free CO/sub 2/) by suspended leaf slices or isolated mesophyll cells from Chenopodium album is increased with increasing pH. Above all, the incorporation of radioactivity into amino acids and malate is stimulated. A direct uptake of HCO/sub 3/ ions and its fixation by PEP carboxylase is suggested. Isolated mesophyll cells showed at pH 7.3 a higher rate of photosynthesis than at pH 5.0.

  17. Incorporation of uridine-H3 into healthy and tobacco necrosis virus-infected mesophyll cells of Chenopodium amaranticolor

    International Nuclear Information System (INIS)

    Faccioli, G.; Rubies-Autonel, C.

    1975-01-01

    Tritiated uridine was selectively incorporated into the nucleus, nucleolus and cytoplasm of actinomycin D-treated Chenopodium amaranticolor cells locally infected with a strain of tobacco necrosis virus (TNV), 3 days after inoculation. Healthy cells did not show such an incorporation. Chloroplasts, in both types of cells, were free of label. Treatment with pancreatic ribonuclease removed the label completely in the majority of nuclei and nucleoli of infected cells. Since infectivity tests showed that AMD treatment increased virus multiplication by 10-12%, it is conceivable to think that the incorporation observed was due to virus synthesis. Preliminary infectivity experiments also showed that treatment of the cells with cycloheximide inhibited virus multiplication up to 80%, while chloramphenicol increased such multiplication. Our results lead to the conclusion that nucleus, nucleolus and cytoplasm but not chloroplasts are the sites involved in the synthesis of TNV. (orig.) [de

  18. Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. A new mechanism for the regulation of stomatal-aperture size in intact leaves: Accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba L.

    Energy Technology Data Exchange (ETDEWEB)

    Lu, P.; Outlaw, W.H. Jr.; Smith, B.G.; Freed, G.A.

    1996-12-31

    At various times after pulse labeling Vicia faba L. leaflets with {sup 14}CO{sub 2}, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents whereas those from rinsed peels contained only cytoplastic contents. Sucrose specific radioactivity peaked in palisade cells, 111 GBq{center_dot}mol{sup {minus}1}, at 20 min. In contrast, the {sup 14}C content and sucrose specific radioactivity were very low in guard cells for 20 min, implying little CO{sub 2} incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum sucrose specific radioactivity and a high sucrose influx rate. These and other comparisons implied the presence of (a) multiple sucrose pools in mesophyll cells, (b) a localized mesophyll-apoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived sucrose in guard-cell walls sufficient to diminish stomatal opening by {approximately} 4 {micro}m. Factors expected to enhance sucrose accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic sucrose concentration, which is elevated when mesophyll-sucrose efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal-aperture size by this previously unrecognized mechanism.

  19. Carbon isotope ratios of epidermal and mesophyll tissues from leaves of C3 and CAM plants

    International Nuclear Information System (INIS)

    Nishida, K.; Roksandic, Z.; Osmond, B.

    1981-01-01

    The δ 13 C values for epidermal and mesophyll tissues of two C 3 plants, Commelina communis and Tulipa gesneriana, and a CAM plant, Kalanchoē daigremontiana, were measured. The values for the tissues of both C 3 plants were similar. In young leaves of Kalanchoē, the epidermis and the mesophyll showed S 13 C values which were nearly identical, and similar to those found in C 3 plants. However, markedly more negative values for epidermal compared to mesophyll tissue, were obtained in the mature Kalanchoē leaf. This is consistent with the facts that the epidermis in a CAM leaf is formed when leaves engage in C 3 photosynthesis and that subsequent dark CO 2 fixation in guard cells or mesophyll cells makes only a small contribution to total epidermal carbon

  20. Single Cell Oncogenesis

    Science.gov (United States)

    Lu, Xin

    It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

  1. Single cell metabolomics

    NARCIS (Netherlands)

    Heinemann, Matthias; Zenobi, Renato

    Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single

  2. Microfluidics for single cell analysis

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant

    Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinical...... problems. In this thesis lab on a chip systems for rare single cell analysis are investigated. The focus was to develop a commercial, disposable device for circulating tumour cell (CTC) analysis. Such a device must be able to separate rare cells from blood samples and subsequently capture the specific...... cells, and simultaneously be fabricated and operated at low costs and be user-friendly. These challenges were addressed through development of two microfluidic devices, one for rare cell isolation based on pinched flow fractionation (PFF) and one for single cell capture based on hydrodynamic trapping...

  3. Single Cell Isolation and Analysis

    Directory of Open Access Journals (Sweden)

    Ping Hu

    2016-10-01

    Full Text Available Increasing evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their peculiar function and fate. Conventional cell based assays mainly analysis the average responses from a population cells, while the difference within individual cells may often be masked. The cell size, RNA transcripts and protein expression level are quite different within individual cells and these variations are key point to answer the problems in cancer, neurobiology, stem cell biology, immunology and developmental biology. To better understand the cell-to-cell variations, the single cell analysis can provide much more detailed information which may be helpful for therapeutic decisions in an increasingly personalized medicine. In this review, we will focus on the recent development in single cell analysis, including methods used in single cell isolation, analysis and some application examples. The review provides the historical background to single cell analysis, discusses limitations, and current and future possibilities in this exciting field of research.

  4. Measuring single-cell density.

    Science.gov (United States)

    Grover, William H; Bryan, Andrea K; Diez-Silva, Monica; Suresh, Subra; Higgins, John M; Manalis, Scott R

    2011-07-05

    We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

  5. The influence of nitric oxide and mercury chloride on leaf mesophyll structure under natural drought conditions

    Directory of Open Access Journals (Sweden)

    Mykola M. Musiyenko

    2012-03-01

    Full Text Available It is established that under natural drought conditions starch was accumulated in the central part of chloroplasts of mesophyll cells and chloroplasts were localized on the periphery of cells at plasmalemma. After treatment wheat plants by nitric oxide donor the decreasing of starch deposits number and close contacts between chloroplasts were indicated, elongated nucleus was localized in the centre of cells. After treatment wheat plant by mercury chloride chloroplasts in the cells lost their oval shape and contacts, increased eventually deposition of starch, indicating the acceleration of aging tissues. Thus, nitric oxide in drought conditions reduced the destructive effect of drought on mesophyll cells, and mercury chloride caused deformation of the membrane cell.

  6. Single-cell photoacoustic thermometry

    Science.gov (United States)

    Gao, Liang; Wang, Lidai; Li, Chiye; Liu, Yan; Ke, Haixin; Zhang, Chi

    2013-01-01

    Abstract. A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community. PMID:23377004

  7. Stomatal conductance, mesophyll conductance, and transpiration efficiency in relation to leaf anatomy in rice and wheat genotypes under drought.

    Science.gov (United States)

    Ouyang, Wenjing; Struik, Paul C; Yin, Xinyou; Yang, Jianchang

    2017-11-02

    Increasing leaf transpiration efficiency (TE) may provide leads for growing rice like dryland cereals such as wheat (Triticum aestivum). To explore avenues for improving TE in rice, variations in stomatal conductance (gs) and mesophyll conductance (gm) and their anatomical determinants were evaluated in two cultivars from each of lowland, aerobic, and upland groups of Oryza sativa, one cultivar of O. glaberrima, and two cultivars of T. aestivum, under three water regimes. The TE of upland rice, O. glaberrima, and wheat was more responsive to the gm/gs ratio than that of lowland and aerobic rice. Overall, the explanatory power of the particular anatomical trait varied among species. Low stomatal density mostly explained the low gs in drought-tolerant rice, whereas rice genotypes with smaller stomata generally responded more strongly to drought. Compared with rice, wheat had a higher gm, which was associated with thicker mesophyll tissue, mesophyll and chloroplasts more exposed to intercellular spaces, and thinner cell walls. Upland rice, O. glaberrima, and wheat cultivars minimized the decrease in gm under drought by maintaining high ratios of chloroplasts to exposed mesophyll cell walls. Rice TE could be improved by increasing the gm/gs ratio via modifying anatomical traits. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

    Science.gov (United States)

    Chiu, Yu-Jui

    To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

  9. Single cell enzyme diagnosis on the chip

    DEFF Research Database (Denmark)

    Jensen, Sissel Juul; Harmsen, Charlotte; Nielsen, Mette Juul

    2013-01-01

    Conventional diagnosis based on ensemble measurements often overlooks the variation among cells. Here, we present a droplet-microfluidics based platform to investigate single cell activities. Adopting a previously developed isothermal rolling circle amplification-based assay, we demonstrate...... detection of enzymatic activities down to the single cell level with small quantities of biological samples, which outcompetes existing techniques. Such a system, capable of resolving single cell activities, will ultimately have clinical applications in diagnosis, prediction of drug response and treatment...

  10. Single-cell sequencing in stem cell biology.

    Science.gov (United States)

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  11. Epigenetics reloaded: the single-cell revolution.

    Science.gov (United States)

    Bheda, Poonam; Schneider, Robert

    2014-11-01

    Mechanistically, how epigenetic states are inherited through cellular divisions remains an important open question in the chromatin field and beyond. Defining the heritability of epigenetic states and the underlying chromatin-based mechanisms within a population of cells is complicated due to cell heterogeneity combined with varying levels of stability of these states; thus, efforts must be focused toward single-cell analyses. The approaches presented here constitute the forefront of epigenetics research at the single-cell level using classic and innovative methods to dissect epigenetics mechanisms from the limited material available in a single cell. This review further outlines exciting future avenues of research to address the significance of epigenetic heterogeneity and the contributions of microfluidics technologies to single-cell isolation and analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Parallel single-cell analysis microfluidic platform

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Gool, Elmar; Frimat, Jean-Philippe; Bomer, Johan G.; van den Berg, Albert; le Gac, Severine

    2011-01-01

    We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed.

  13. Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange.

    Science.gov (United States)

    Dow, Graham J; Berry, Joseph A; Bergmann, Dominique C

    2017-10-01

    Stomata are simultaneously tasked with permitting the uptake of carbon dioxide for photosynthesis while limiting water loss from the plant. This process is mainly regulated by guard cell control of the stomatal aperture, but recent advancements have highlighted the importance of several genes that control stomatal development. Using targeted genetic manipulations of the stomatal lineage and a combination of gas exchange and microscopy techniques, we show that changes in stomatal development of the epidermal layer lead to coupled changes in the underlying mesophyll tissues. This coordinated response tends to match leaf photosynthetic potential (V cmax ) with gas-exchange capacity (g smax ), and hence the uptake of carbon dioxide for water lost. We found that different genetic regulators systematically altered tissue coordination in separate ways: the transcription factor SPEECHLESS (SPCH) primarily affected leaf size and thickness, whereas peptides in the EPIDERMAL PATTERNING FACTOR (EPF) family altered cell density in the mesophyll. It was also determined that interlayer coordination required the cell-surface receptor TOO MANY MOUTHS (TMM). These results demonstrate that stomata-specific regulators can alter mesophyll properties, which provides insight into how molecular pathways can organize leaf tissues to coordinate gas exchange and suggests new strategies for improving plant water-use efficiency. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas; Gojobori, Takashi

    2015-01-01

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  15. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas

    2015-10-22

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  16. Automated Single Cell Data Decontamination Pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Tennessen, Kristin [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.; Pati, Amrita [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.

    2014-03-21

    Recent technological advancements in single-cell genomics have encouraged the classification and functional assessment of microorganisms from a wide span of the biospheres phylogeny.1,2 Environmental processes of interest to the DOE, such as bioremediation and carbon cycling, can be elucidated through the genomic lens of these unculturable microbes. However, contamination can occur at various stages of the single-cell sequencing process. Contaminated data can lead to wasted time and effort on meaningless analyses, inaccurate or erroneous conclusions, and pollution of public databases. A fully automated decontamination tool is necessary to prevent these instances and increase the throughput of the single-cell sequencing process

  17. Infection of potato mesophyll protoplasts with five plant viruses.

    Science.gov (United States)

    Barker, H; Harrison, B D

    1982-12-01

    Methods are described for preparing potato mesophyll protoplasts that are suitable for infection with inocula of virus nucleoprotein or RNA. The protoplasts could be infected with four sap-transmissible viruses (tobacco mosaic, tobacco rattle, tobacco ringspot and tomato black ring viruses) and with potato leafroll virus, which is not saptransmissible. No differences were observed in ability to infect protoplasts with potato leafroll virus strains differing either in virulence in intact plants or in aphid transmissibility.

  18. Technologies for Single-Cell Isolation.

    Science.gov (United States)

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-07-24

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

  19. Technologies for Single-Cell Isolation

    Science.gov (United States)

    Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

    2015-01-01

    The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

  20. Technologies for Single-Cell Isolation

    Directory of Open Access Journals (Sweden)

    Andre Gross

    2015-07-01

    Full Text Available The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting respectively Flow cytometry (33% usage, laser microdissection (17%, manual cell picking (17%, random seeding/dilution (15%, and microfluidics/lab-on-a-chip devices (12% are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

  1. Single-cell measurement of red blood cell oxygen affinity

    OpenAIRE

    Caprio, Di; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

    2015-01-01

    Oxygen is transported throughout the body by hemoglobin in red blood cells. While the oxygen affinity of blood is well understood and is routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of red blood cell volume and hemoglobin concentration are taken millions of times per day by clinical hematology analyzers and are important factors in determining the health of the hematologic system....

  2. Single cell elemental analysis using nuclear microscopy

    International Nuclear Information System (INIS)

    Ren, M.Q.; Thong, P.S.P.; Kara, U.; Watt, F.

    1999-01-01

    The use of Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) to provide quantitative elemental analysis of single cells is an area which has high potential, particularly when the trace elements such as Ca, Fe, Zn and Cu can be monitored. We describe the methodology of sample preparation for two cell types, the procedures of cell imaging using STIM, and the quantitative elemental analysis of single cells using RBS and PIXE. Recent work on single cells at the Nuclear Microscopy Research Centre,National University of Singapore has centred around two research areas: (a) Apoptosis (programmed cell death), which has been recently implicated in a wide range of pathological conditions such as cancer, Parkinson's disease etc, and (b) Malaria (infection of red blood cells by the malaria parasite). Firstly we present results on the elemental analysis of human Chang liver cells (ATTCC CCL 13) where vanadium ions were used to trigger apoptosis, and demonstrate that nuclear microscopy has the capability of monitoring vanadium loading within individual cells. Secondly we present the results of elemental changes taking place in individual mouse red blood cells which have been infected with the malaria parasite and treated with the anti-malaria drug Qinghaosu (QHS)

  3. New frontiers in single-cell analysis

    OpenAIRE

    Templer, Richard H.; Ces, Oscar

    2008-01-01

    For this special issue of J. R. Soc. Interface we present an overview of the driving forces behind technological advances in the field of single-cell analysis. These range from increasing our understanding of cellular heterogeneity through to the study of rare cells, areas of research that cannot be tackled effectively using current high-throughput population-based averaging techniques.

  4. Experimental techniques for single cell and single molecule biomechanics

    International Nuclear Information System (INIS)

    Lim, C.T.; Zhou, E.H.; Li, A.; Vedula, S.R.K.; Fu, H.X.

    2006-01-01

    Stresses and strains that act on the human body can arise either from external physical forces or internal physiological environmental conditions. These biophysical interactions can occur not only at the musculoskeletal but also cellular and molecular levels and can determine the health and function of the human body. Here, we seek to investigate the structure-property-function relationship of cells and biomolecules so as to understand their important physiological functions as well as establish possible connections to human diseases. With the recent advancements in cell and molecular biology, biophysics and nanotechnology, several innovative and state-of-the-art experimental techniques and equipment have been developed to probe the structural and mechanical properties of biostructures from the micro- down to picoscale. Some of these experimental techniques include the optical or laser trap method, micropipette aspiration, step-pressure technique, atomic force microscopy and molecular force spectroscopy. In this article, we will review the basic principles and usage of these techniques to conduct single cell and single molecule biomechanics research

  5. Single Nanowire Probe for Single Cell Endoscopy and Sensing

    Science.gov (United States)

    Yan, Ruoxue

    The ability to manipulate light in subwavelength photonic and plasmonic structures has shown great potentials in revolutionizing how information is generated, transformed and processed. Chemically synthesized nanowires, in particular, offers a unique toolbox not only for highly compact and integrated photonic modules and devices, including coherent and incoherent light sources, waveguides, photodetectors and photovoltaics, but also for new types of nanoscopic bio-probes for spot cargo delivery and in-situ single cell endoscopy and sensing. Such nanowire probes would enable us to carry out intracellular imaging and probing with high spatial resolution, monitor in-vivo biological processes within single living cells and greatly improve our fundamental understanding of cell functions, intracellular physiological processes, and cellular signal pathways. My work is aimed at developing a material and instrumental platform for such single nanowire probe. Successful optical integration of Ag nanowire plasmonic waveguides, which offers deep subwavelength mode confinement, and conventional photonic waveguides was demonstrated on a single nanowire level. The highest plasmonic-photonic coupling efficiency coupling was found at small coupling angles and low input frequencies. The frequency dependent propagation loss was observed in Ag nanowire and was confirmed by quantitative measurement and in agreement with theoretical expectations. Rational integration of dielectric and Ag nanowire waveguide components into hybrid optical-plasmonic routing devices has been demonstrated. This capability is essential for incorporating sub-100nm Ag nanowire waveguides into optical fiber based nanoprobes for single cell endoscopy. The nanoprobe system based on single nanowire waveguides was demonstrated by optically coupling semiconductor or metal nanowire with an optical fiber with tapered tip. This nanoprobe design requires minimal instrumentation which makes it cost efficient and readily

  6. Single-cell measurement of red blood cell oxygen affinity.

    Science.gov (United States)

    Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

    2015-08-11

    Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

  7. Capillary Electrophoretic Technologies for Single Cell Metabolomics

    Science.gov (United States)

    Lapainis, Theodore E.

    2009-01-01

    Understanding the functioning of the brain is hindered by a lack of knowledge of the full complement of neurotransmitters and neuromodulatory compounds. Single cell measurements aid in the discovery of neurotransmitters used by small subsets of neurons that would be diluted below detection limits or masked by ubiquitous compounds when working with…

  8. Assessing T cell differentiation at the single-cell level

    NARCIS (Netherlands)

    Gerlach, Carmen

    2012-01-01

    This thesis describes the development and use of a novel technology for single-cell fate mapping, called cellular barcoding. With this technology, unique and heritable genetic tags (barcodes) are introduced into naïve T cells. Using cellular barcoding, we investigated I) how different

  9. Single-Cell RNA Sequencing of Glioblastoma Cells.

    Science.gov (United States)

    Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

    2018-01-01

    Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

  10. Dissecting stem cell differentiation using single cell expression profiling

    OpenAIRE

    Moignard, Victoria Rachel; Göttgens, Berthold

    2016-01-01

    Many assumptions about the way cells behave are based on analyses of populations. However, it is now widely recognized that even apparently pure populations can display a remarkable level of heterogeneity. This is particularly true in stem cell biology where it hinders our understanding of normal development and the development of strategies for regenerative medicine. Over the past decade technologies facilitating gene expression analysis at the single cell level have become widespread, provi...

  11. Interaction of E. coli DNA with tobacco mesophyll protoplasts

    International Nuclear Information System (INIS)

    Heyn, R.F.

    1975-01-01

    This chapter is part of a dissertation dealing with the interaction of DNA with protoplasts. Having established the length of time during which tobacco mesophyll protoplasts do not synthesize DNA following their isolation, it is important to know the extent of DNA uptake just before the onset of DNA synthesis (and possible integration) and to find optimal conditions for this uptake. Therefore, the association of E. coli DNA with tobacco protoplasts was studied. Care should be taken with the interpretation of ''uptake'' results: adsorption phenomena play a very important role and may do so at the plasmalemma of naked protoplasts. To solve the problems involved, the use of radiation-damaged DNA was attempted. With E. coli DNA possessing a large number of thymine containing pyrimidine dimers, the loss of dimers from DNA recovered from treated protoplasts was tested in order to obtain an indication of ''real'' uptake. The results are reported

  12. Estimation of mesophyll conductance to CO2 flux by three different methods

    International Nuclear Information System (INIS)

    Loreto, F.; Harley, P.C.; Di Marco, G.; Sharkey, T.D.

    1992-01-01

    The resistance to diffusion of CO2 from the intercellular airspaces within the leaf through the mesophyll to the sites of carboxylation during photosynthesis was measured using three different techniques, The three techniques include a method based on discrimination against the heavy stable isotope of carbon, 13C, and two modeling methods. The methods rely upon different assumptions, but the estimates of mesophyll conductance were similar with all three methods. The mesophyll conductance of leaves from a number of species was about 1.4 times the stomatal conductance for CO2 diffusion determined in unstressed plants at high light. The relatively low CO2 partial pressure inside chloroplasts of plants with a low mesophyll conductance did not lead to enhanced O2 sensitivity of photosynthesis because the low conductance caused a significant drop in the chloroplast CO2 Partial pressure upon switching to low O2. We found no correlation between mesophyll conductance and the ratio of internal leaf area to leaf surface area and only a weak correlation between mesophyll conductance and the proportion of leaf volume occupied by air. Mesophyll conductance was independent of CO2 and O2 partial pressure during the measurement, indicating that a true physical parameter, independent of biochemical effects, was being measured. No evidence for accumulating mechanisms was found. Some plants, notably Citrus aurantium and Simmondsia chinensis, had very low conductances that limit the rate of photosynthesis these plants can attain at atmospheric CO2 level

  13. Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis.

    Science.gov (United States)

    Wigoda, Noa; Pasmanik-Chor, Metsada; Yang, Tianyuan; Yu, Ling; Moshelion, Menachem; Moran, Nava

    2017-06-01

    Under fluctuating ambient conditions, the ability of plants to maintain hydromineral homeostasis requires the tight control of long distance transport. This includes the control of radial transport within leaves, from veins to mesophyll. The bundle sheath is a structure that tightly wraps around leaf vasculature. It has been suggested to act as a selective barrier in the context of radial transport. This suggestion is based on recent physiological transport assays of bundle sheath cells (BSCs), as well as the anatomy of these cells.We hypothesized that the unique transport functionality of BSCs is apparent in their transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) from the leaves of Arabidopsis thaliana. Of the 90 genes differentially expressed between BSCs and MCs, 45% are membrane related and 20% transport related, a prominent example being the proton pump AHA2. Electrophysiological assays showed that the major AKT2-like membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Taken together, these differences may cause simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs, in otherwise similar conditions. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  14. Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

    Czech Academy of Sciences Publication Activity Database

    Albrechtová, Jana; Janáček, Jiří; Lhotáková, Zuzana; Radochová, Barbora; Kubínová, Lucie

    2007-01-01

    Roč. 58, č. 6 (2007), s. 1451-1461 ISSN 0022-0957 R&D Projects: GA AV ČR IAA5011810; GA AV ČR(CZ) IAA600110507; GA MŠk(CZ) LC06063 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z60050516 Keywords : mesophyll * stereology * confocal microscopy Subject RIV: EA - Cell Biology Impact factor: 3.917, year: 2007

  15. Gravisensing in single-celled systems

    Science.gov (United States)

    Braun, M.; Limbach, C.

    Single-celled systems are favourable cell types for studying several aspects of gravisensing and gravitropic responses. Whether and how actin is involved in both processes in higher plant statocytes is still a matter of intensive debate. In single-celled and tip-growing characean rhizoids and protonemata, however, there is clear evidence that actin is a central keyplayer controlling polarized growth and the mechanisms of gravity sensing and growth reorientation. Both cell types exhibit a unique actin polymerization in the extending tip, strictly colocalized with the prominent ER-aggregate in the center of the Spitzenkoerper. The local accumulation of ADF and profilin in this central array suggest that actin polymerization is controlled by these actin-binding proteins, which can be regulated by calcium, pH and a variety of other parameters. Distinct actin filaments extend even into the outermost tip and form a dense meshwork in the apical and subapical region, before they become bundled by villin to form two populations of thick actin cables that generate rotational cytoplasmic streaming in the basal region. Actomyosin not only mediates the delivery of secretory vesicles to the growing tip and controls the incorporation pattern of cell wall material, but also coordinates the tip-focused distribution pattern of calcium channels in the apical membrane. They establish the tip-high calcium gradient, a prerequisite for exocytosis. Microgravity experiments have added much to our understanding that both cell types use an efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. Actin's involvement in the graviresponses is more indirect. The upward growth of negatively gravitropic protonemata was shown to be preceded by a statolith-induced relocalization the Ca2+-calcium gradient to the upper flank that does not occur in positively gravitropic

  16. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

    Science.gov (United States)

    Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

    2011-01-01

    The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis 60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

  17. Biological Evaluation of Single Cell Protein

    International Nuclear Information System (INIS)

    Hasan, I.A.; Mohamed, N.E.; El-Sayed, E.A.; Younis, N.A.

    2011-01-01

    In this study, the nutritional value of single cell protein (SCP) was evaluated as a non conventional protein source produced by fermenting fungal local strains of Trichoderma longibrachiatum, Aspergillus niger, Aspergillus terreus and Penicillium funiculosum with alkali treated sugar cane bagasse. Amino acid analysis revealed that the produced SCP contains essential and non essential amino acids. Male mice were fed on normal (basal) diet which contains 18% conventional protein and served as control group. In the second (T1) and the third (T2) group, the animals were fed on a diet in which 15% and 30% of conventional protein source were replaced by SCP, respectively. At intervals of 15, 30, 45 and 60 days, mice were sacrificed and the blood samples were collected for the biochemical evaluation. The daily averages of body weight were significantly higher with group T2 than group T1. Where as, the kidney weights in groups (T1) and (T2) were significantly increased as compared with control. A non significant difference between the tested groups in the enzyme activities of AST, ALT and GSH content of liver tissue were recorded. While, cholesterol and triglycerides contents showed a significant decrease in both (T1) and (T2) groups as compared with control. The recorded values of the serum hormone (T4), ALP activities, albumin and A/G ratio did not changed by the previous treatments. Serum levels of total protein, urea, creatinine and uric acid were higher for groups (T1) and (T2) than the control group. In conclusion, partial substitution of soy bean protein in mice diet with single cell protein (15%) improved the mice growth without any adverse effects on some of the physiological functions tested

  18. Micro-PIXE for single cell analysis

    International Nuclear Information System (INIS)

    Ortega, Richard

    2012-01-01

    The knowledge of the intracellular distribution of biological relevant metals is important to understand their mechanisms of action in cells, either for physiological, toxicological or pathological processes. However, the direct detection of trace metals in single cells is a challenging task that requires sophisticated analytical developments. The combination of micro-PIXE with RBS and STIM (Scanning Transmission Ion Microscopy) allows the quantitative determination of trace metal content within sub-cellular compartments. The application of STIM analysis provides high spatial resolution imaging (< 200 nm) and excellent mass sensitivity (< 0.1 ng). Application of the STIM-PIXE-RBS methodology is absolutely needed when organic mass loss appears during PIXE-RBS irradiation. This combination of STIM-PIXE-RBS provides fully quantitative determination of trace element content, expressed in μg/g, which is a quite unique capability for micro-PIXE compared to other micro-analytical methods such as the electron and synchrotron x-ray fluorescence. Examples of micro-PIXE studies for sub-cellular imaging of trace elements in various fields of interest will be presented: in patho-physiology of trace elements involved in neurodegenerative diseases such as Parkinson's disease, and in toxicology of metals such as cobalt. (author)

  19. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level. Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining...... on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time...

  20. Single cell protein from mandarin orange peel

    Energy Technology Data Exchange (ETDEWEB)

    Mishio, M.; Magai, J.

    1981-01-01

    As the hydrolysis of mandarin orange peel with macerating enzyme (40 degrees C, 24 h) produced 0.59 g g-1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120 degrees C with 0.8 N H2S04), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g-1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30 degrees C using 100g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts. (Refs. 12).

  1. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation

    Science.gov (United States)

    Bogdanov, Evgeny; Dominova, Irina; Shusharina, Natalia; Botman, Stepan; Kasymov, Vitaliy; Patrushev, Maksim

    2013-01-01

    A limited amount of DNA extracted from single cells, and the development of single cell diagnostics make it necessary to create a new highly effective method for the single cells nucleic acids isolation. In this paper, we propose the DNA isolation method from biomaterials with limited DNA quantity in sample, and from samples with degradable DNA based on the use of solid-phase adsorbent silicon dioxide nanofilm deposited on the inner surface of PCR tube. PMID:23874571

  2. Proton extrusion is an essential signalling component in the HR of epidermal single cells in the barley-powdery mildew interaction

    DEFF Research Database (Denmark)

    Zhou, F.S.; Andersen, C.H.; Burhenne, K.

    2000-01-01

    We propose a model for activation of the epidermal cell hypersensitive response (HR) in the barley/powdery mildew interaction. The model suggests that the plasma membrane proton pump (H+-ATPase) of epidermal cells is activated following penetration by an avirulent powdery mildew fungus...... in the incompatible interaction; (4) race-specific proton extrusion is observed underneath epidermal tissue detached from leaves inoculated 15 h earlier; and (5) treatment of leaves with fusicoccin, an activator of the plasma membrane H+-ATPase, increases the number of HR-cells in the compatible interaction........ This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR-related H2O2 is found in the attachment zones...

  3. High-dimensional single-cell cancer biology.

    Science.gov (United States)

    Irish, Jonathan M; Doxie, Deon B

    2014-01-01

    Cancer cells are distinguished from each other and from healthy cells by features that drive clonal evolution and therapy resistance. New advances in high-dimensional flow cytometry make it possible to systematically measure mechanisms of tumor initiation, progression, and therapy resistance on millions of cells from human tumors. Here we describe flow cytometry techniques that enable a "single-cell " view of cancer. High-dimensional techniques like mass cytometry enable multiplexed single-cell analysis of cell identity, clinical biomarkers, signaling network phospho-proteins, transcription factors, and functional readouts of proliferation, cell cycle status, and apoptosis. This capability pairs well with a signaling profiles approach that dissects mechanism by systematically perturbing and measuring many nodes in a signaling network. Single-cell approaches enable study of cellular heterogeneity of primary tissues and turn cell subsets into experimental controls or opportunities for new discovery. Rare populations of stem cells or therapy-resistant cancer cells can be identified and compared to other types of cells within the same sample. In the long term, these techniques will enable tracking of minimal residual disease (MRD) and disease progression. By better understanding biological systems that control development and cell-cell interactions in healthy and diseased contexts, we can learn to program cells to become therapeutic agents or target malignant signaling events to specifically kill cancer cells. Single-cell approaches that provide deep insight into cell signaling and fate decisions will be critical to optimizing the next generation of cancer treatments combining targeted approaches and immunotherapy.

  4. Application of single-cell technology in cancer research.

    Science.gov (United States)

    Liang, Shao-Bo; Fu, Li-Wu

    2017-07-01

    In this review, we have outlined the application of single-cell technology in cancer research. Single-cell technology has made encouraging progress in recent years and now provides the means to detect rare cancer cells such as circulating tumor cells and cancer stem cells. We reveal how this technology has advanced the analysis of intratumor heterogeneity and tumor epigenetics, and guided individualized treatment strategies. The future prospects now are to bring single-cell technology into the clinical arena. We believe that the clinical application of single-cell technology will be beneficial in cancer diagnostics and treatment, and ultimately improve survival in cancer patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Biology at a single cell level

    CSIR Research Space (South Africa)

    Mthunzi, P

    2012-10-01

    Full Text Available ://www.regenexx.com/wp-content/uploads/2011/05/IPS-cell-problems.jpg Induced pluripotent stem cells differentiated in culture http://www.youtube.com/watch?v=ECllrIzTKbA&feature=related Transfecting neuroblastomas Neuroblastoma ? Brain cells ? 80 ? 120 billion neurons in human... brain ? Non- renewing cell type ? Neurons difficult to transfect with established protocols ? Susceptible to degenerative disorders: - Parkinson?s disease - Multiple sclerosis - Alzheimer's disease http...

  6. Going single but not solo with podocytes: potentials, limitations, and pitfalls of single-cell analysis.

    Science.gov (United States)

    Schiffer, Mario

    2017-11-01

    Single-cell RNA-sequence (RNA-seq) is a widely used tool to study biological questions in single cells. The discussed study identified 92 genes being predominantly expressed in podocytes based on a 5-fold higher expression compared with endothelial and mesangial cells. In addition to technical pitfalls, the question that is discussed in this commentary is whether results of a single-cell RNAseq study are able to deliver expression data that truly characterize a podocyte. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  7. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  8. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  9. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  10. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  11. Single-cell technologies to study the immune system.

    Science.gov (United States)

    Proserpio, Valentina; Mahata, Bidesh

    2016-02-01

    The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

  12. Single Cell Genomics: Approaches and Utility in Immunology

    Science.gov (United States)

    Neu, Karlynn E; Tang, Qingming; Wilson, Patrick C; Khan, Aly A

    2017-01-01

    Single cell genomics offers powerful tools for studying lymphocytes, which make it possible to observe rare and intermediate cell states that cannot be resolved at the population-level. Advances in computer science and single cell sequencing technology have created a data-driven revolution in immunology. The challenge for immunologists is to harness computing and turn an avalanche of quantitative data into meaningful discovery of immunological principles, predictive models, and strategies for therapeutics. Here, we review the current literature on computational analysis of single cell RNA-seq data and discuss underlying assumptions, methods, and applications in immunology, and highlight important directions for future research. PMID:28094102

  13. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

    Science.gov (United States)

    Eustaquio, Trisha; Leary, James F

    2012-01-01

    Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

  14. Multispectral optical tweezers for molecular diagnostics of single biological cells

    Science.gov (United States)

    Butler, Corey; Fardad, Shima; Sincore, Alex; Vangheluwe, Marie; Baudelet, Matthieu; Richardson, Martin

    2012-03-01

    Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.

  15. Using Single-Protein Tracking to Study Cell Migration.

    Science.gov (United States)

    Orré, Thomas; Mehidi, Amine; Massou, Sophie; Rossier, Olivier; Giannone, Grégory

    2018-01-01

    To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

  16. Measurement of gross photosynthesis, respiration in the light, and mesophyll conductance using H218O labeling.

    Science.gov (United States)

    Gauthier, Paul Pg; Battle, Mark O; Griffin, Kevin L; Bender, Michael L

    2018-03-27

    A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesis and O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas exchange measurement. The petiole is immersed in water enriched to a δ18O of ~9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2. GOP is calculated from the increase in δ18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2. Both terms are measured by isotope ratio mass spectrometry. CO2 assimilation and other standard gas exchange parameters are also measured. Reproducible measurements are made on a single leaf for more than 15 hours. We used this method to measure the light response curve of NOP and GOP in Phaseolus vulgaris at 21% and 2% O2. We then used these data to examine the O2/CO2 ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

  17. Mechanical control of mitotic progression in single animal cells

    OpenAIRE

    Cattin, Cedric J.; Düggelin, Marcel; Martinez-Martin, David; Gerber, Christoph; Müller, Daniel J.; Stewart, Martin P.

    2015-01-01

    Despite the importance of mitotic cell rounding in tissue development and cell proliferation, there remains a paucity of approaches to investigate the mechanical robustness of cell rounding. Here we introduce ion beam-sculpted microcantilevers that enable precise force-feedback-controlled confinement of single cells while characterizing their progression through mitosis. We identify three force regimes according to the cell response: small forces (∼5 nN) that accelerate mitotic progression, i...

  18. Single cell analysis of normal and leukemic hematopoiesis.

    Science.gov (United States)

    Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

    2018-02-01

    The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

  19. Bioinformatics approaches to single-cell analysis in developmental biology.

    Science.gov (United States)

    Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

    2016-03-01

    Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European

  20. Single-cell Analysis of Lambda Immunity Regulation

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Svenningsen, Sine Lo; Eisen, Harvey

    2003-01-01

    We have examined expression of the ¿cI operon in single cells via a rexgfp substitution. Although average fluorescence agreed with expectations for expression of ¿-repressor, fluorescence fluctuated greatly from cell-to-cell. Fluctuations in repressor concentration are not predicted by previous m...

  1. Tip chip : Subcellular sampling from single cancer cells

    NARCIS (Netherlands)

    Quist, Jos; Sarajlic, Edin; Lai, Stanley C.S.; Lemay, Serge G.

    2016-01-01

    To analyze the molecular content of single cells, cell lysis is typically required, yielding a snapshot of cell behavior only. To follow complex molecular profiles over time, subcellular sampling methods potentially can be used, but to date these methods involve laborious offline analysis. Here we

  2. Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Erica L Carpenter

    2014-07-01

    Full Text Available Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

  3. Single-hit mechanism of tumour cell killing by radiation.

    Science.gov (United States)

    Chapman, J D

    2003-02-01

    To review the relative importance of the single-hit mechanism of radiation killing for tumour response to 1.8-2.0 Gy day(-1) fractions and to low dose-rate brachytherapy. Tumour cell killing by ionizing radiation is well described by the linear-quadratic equation that contains two independent components distinguished by dose kinetics. Analyses of tumour cell survival curves that contain six or more dose points usually provide good estimates of the alpha- and beta-inactivation coefficients. Superior estimates of tumour cell intrinsic radiosensitivity are obtained when synchronized populations are employed. The characteristics of single-hit inactivation of tumour cells are reviewed and compared with the characteristics of beta-inactivation. Potential molecular targets associated with single-hit inactivation are discussed along with strategies for potentiating cell killing by this mechanism. The single-hit mechanism of tumour cell killing shows no dependence on dose-rate and, consequently, no evidence of sublethal damage repair. It is uniquely potentiated by high linear-energy-transfer radiation, exhibits a smaller oxygen enhancement ratio and exhibits a larger indirect effect by hydroxyl radicals than the beta-mechanism. alpha-inactivation coefficients vary slightly throughout interphase but mitotic cells exhibit extremely high alpha-coefficients in the range of those observed for lymphocytes and some repair-deficient cells. Evidence is accumulating to suggest that chromatin in compacted form could be a radiation-hypersensitive target associated with single-hit radiation killing. Analyses of tumour cell survival curves demonstrate that it is the single-hit mechanism (alpha) that determines the majority of cell killing after doses of 2Gy and that this mechanism is highly variable between tumour cell lines. The characteristics of single-hit inactivation are qualitatively and quantitatively distinct from those of beta-inactivation. Compacted chromatin in tumour cells

  4. Quantitative high-resolution genomic analysis of single cancer cells.

    Science.gov (United States)

    Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

    2011-01-01

    During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  5. Quantitative high-resolution genomic analysis of single cancer cells.

    Directory of Open Access Journals (Sweden)

    Juliane Hannemann

    Full Text Available During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

  6. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  7. Single-cell regulome data analysis by SCRAT.

    Science.gov (United States)

    Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

    2017-09-15

    Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  8. Platforms for Single-Cell Collection and Analysis

    Directory of Open Access Journals (Sweden)

    Lukas Valihrach

    2018-03-01

    Full Text Available Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS. In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  9. Platforms for Single-Cell Collection and Analysis.

    Science.gov (United States)

    Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

    2018-03-11

    Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  10. Micropillar arrays enabling single microbial cell encapsulation in hydrogels.

    Science.gov (United States)

    Park, Kyun Joo; Lee, Kyoung G; Seok, Seunghwan; Choi, Bong Gill; Lee, Moon-Keun; Park, Tae Jung; Park, Jung Youn; Kim, Do Hyun; Lee, Seok Jae

    2014-06-07

    Single microbial cell encapsulation in hydrogels is an important task to find valuable biological resources for human welfare. The conventional microfluidic designs are mainly targeted only for highly dispersed spherical bioparticles. Advanced structures should be taken into consideration for handling such aggregated and non-spherical microorganisms. Here, to address the challenge, we propose a new type of cylindrical-shaped micropillar array in a microfluidic device for enhancing the dispersion of cell clusters and the isolation of individual cells into individual micro-hydrogels for potential practical applications. The incorporated micropillars act as a sieve for the breaking of Escherichia coli (E. coli) clusters into single cells in a polymer mixture. Furthermore, the combination of hydrodynamic forces and a flow-focusing technique will improve the probability of encapsulation of a single cell into each hydrogel with a broad range of cell concentrations. This proposed strategy and device would be a useful platform for genetically modified microorganisms for practical applications.

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Single-cell intracellular nano-pH probes†

    OpenAIRE

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

  13. Single-cell magnetic imaging using a quantum diamond microscope.

    Science.gov (United States)

    Glenn, D R; Lee, K; Park, H; Weissleder, R; Yacoby, A; Lukin, M D; Lee, H; Walsworth, R L; Connolly, C B

    2015-08-01

    We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

  14. Solar cell structure incorporating a novel single crystal silicon material

    Science.gov (United States)

    Pankove, Jacques I.; Wu, Chung P.

    1983-01-01

    A novel hydrogen rich single crystal silicon material having a band gap energy greater than 1.1 eV can be fabricated by forming an amorphous region of graded crystallinity in a body of single crystalline silicon and thereafter contacting the region with atomic hydrogen followed by pulsed laser annealing at a sufficient power and for a sufficient duration to recrystallize the region into single crystal silicon without out-gassing the hydrogen. The new material can be used to fabricate semiconductor devices such as single crystal silicon solar cells with surface window regions having a greater band gap energy than that of single crystal silicon without hydrogen.

  15. Plant Systems Biology at the Single-Cell Level.

    Science.gov (United States)

    Libault, Marc; Pingault, Lise; Zogli, Prince; Schiefelbein, John

    2017-11-01

    Our understanding of plant biology is increasingly being built upon studies using 'omics and system biology approaches performed at the level of the entire plant, organ, or tissue. Although these approaches open new avenues to better understand plant biology, they suffer from the cellular complexity of the analyzed sample. Recent methodological advances now allow plant scientists to overcome this limitation and enable biological analyses of single-cells or single-cell-types. Coupled with the development of bioinformatics and functional genomics resources, these studies provide opportunities for high-resolution systems analyses of plant phenomena. In this review, we describe the recent advances, current challenges, and future directions in exploring the biology of single-cells and single-cell-types to enhance our understanding of plant biology as a system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Functional Insights into Sponge Microbiology by Single Cell Genomics

    KAUST Repository

    Hentschel, Ute

    2011-04-09

    Marine Sponges (Porifera) are known to harbor enormous amounts of microorganisms with members belonging to at least 30 different bacterial phyla including several candidate phyla and both archaeal lineages. Here, we applied single cell genomics to the mic

  17. Sampling strategies to capture single-cell heterogeneity

    OpenAIRE

    Satwik Rajaram; Louise E. Heinrich; John D. Gordan; Jayant Avva; Kathy M. Bonness; Agnieszka K. Witkiewicz; James S. Malter; Chloe E. Atreya; Robert S. Warren; Lani F. Wu; Steven J. Altschuler

    2017-01-01

    Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here, we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples. ...

  18. Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

    Science.gov (United States)

    Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

    2016-05-01

    Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

  19. Single-Cell Genomics: Approaches and Utility in Immunology.

    Science.gov (United States)

    Neu, Karlynn E; Tang, Qingming; Wilson, Patrick C; Khan, Aly A

    2017-02-01

    Single-cell genomics offers powerful tools for studying immune cells, which make it possible to observe rare and intermediate cell states that cannot be resolved at the population level. Advances in computer science and single-cell sequencing technology have created a data-driven revolution in immunology. The challenge for immunologists is to harness computing and turn an avalanche of quantitative data into meaningful discovery of immunological principles, predictive models, and strategies for therapeutics. Here, we review the current literature on computational analysis of single-cell RNA-sequencing data and discuss underlying assumptions, methods, and applications in immunology, and highlight important directions for future research. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Clustering Single-Cell Expression Data Using Random Forest Graphs.

    Science.gov (United States)

    Pouyan, Maziyar Baran; Nourani, Mehrdad

    2017-07-01

    Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.

  1. Single cell transcriptome profiling of developing chick retinal cells.

    Science.gov (United States)

    Laboissonniere, Lauren A; Martin, Gregory M; Goetz, Jillian J; Bi, Ran; Pope, Brock; Weinand, Kallie; Ellson, Laura; Fru, Diane; Lee, Miranda; Wester, Andrea K; Liu, Peng; Trimarchi, Jeffrey M

    2017-08-15

    The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types. © 2017 Wiley Periodicals, Inc.

  2. Single-cell intracellular nano-pH probes†

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2016-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

  3. Single-cell intracellular nano-pH probes.

    Science.gov (United States)

    Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

    2015-01-01

    Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

  4. Parameter Screening in Microfluidics Based Hydrodynamic Single-Cell Trapping

    Directory of Open Access Journals (Sweden)

    B. Deng

    2014-01-01

    Full Text Available Microfluidic cell-based arraying technology is widely used in the field of single-cell analysis. However, among developed devices, there is a compromise between cellular loading efficiencies and trapped cell densities, which deserves further analysis and optimization. To address this issue, the cell trapping efficiency of a microfluidic device with two parallel micro channels interconnected with cellular trapping sites was studied in this paper. By regulating channel inlet and outlet status, the microfluidic trapping structure can mimic key functioning units of previously reported devices. Numerical simulations were used to model this cellular trapping structure, quantifying the effects of channel on/off status and trapping structure geometries on the cellular trapping efficiency. Furthermore, the microfluidic device was fabricated based on conventional microfabrication and the cellular trapping efficiency was quantified in experiments. Experimental results showed that, besides geometry parameters, cellular travelling velocities and sizes also affected the single-cell trapping efficiency. By fine tuning parameters, more than 95% of trapping sites were taken by individual cells. This study may lay foundation in further studies of single-cell positioning in microfluidics and push forward the study of single-cell analysis.

  5. The single-cell gel electrophoresis assay to determine apoptosis ...

    African Journals Online (AJOL)

    When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

  6. Single cell adhesion assay using computer controlled micropipette.

    Directory of Open Access Journals (Sweden)

    Rita Salánki

    Full Text Available Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day. Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min. We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a

  7. Mapping Cellular Hierarchy by Single-Cell Analysis of the Cell Surface Repertoire

    OpenAIRE

    Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A.; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

    2013-01-01

    Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insi...

  8. Scientist, Single Cell Analysis Facility | Center for Cancer Research

    Science.gov (United States)

    The Cancer Research Technology Program (CRTP) develops and implements emerging technology, cancer biology expertise and research capabilities to accomplish NCI research objectives.  The CRTP is an outward-facing, multi-disciplinary hub purposed to enable the external cancer research community and provides dedicated support to NCI’s intramural Center for Cancer Research (CCR).  The dedicated units provide electron microscopy, protein characterization, protein expression, optical microscopy and nextGen sequencing. These research efforts are an integral part of CCR at the Frederick National Laboratory for Cancer Research (FNLCR).  CRTP scientists also work collaboratively with intramural NCI investigators to provide research technologies and expertise. KEY ROLES AND RESPONSIBILITIES We are seeking a highly motivated Scientist II to join the newly established Single Cell Analysis Facility (SCAF) of the Center for Cancer Research (CCR) at NCI. The SCAF will house state of the art single cell sequencing technologies including 10xGenomics Chromium, BD Genomics Rhapsody, DEPPArray, and other emerging single cell technologies. The Scientist: Will interact with close to 200 laboratories within the CCR to design and carry out single cell experiments for cancer research Will work on single cell isolation/preparation from various tissues and cells and related NexGen sequencing library preparation Is expected to author publications in peer reviewed scientific journals

  9. Addressable droplet microarrays for single cell protein analysis.

    Science.gov (United States)

    Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

    2014-11-07

    Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

  10. Optical and hydrodynamic stretching of single cells from blood

    DEFF Research Database (Denmark)

    Thirstrup, Henrik; Rungling, Tony B.; Khalil Al-Hamdani, Mustafa Zyad

    2017-01-01

    Mechanical properties, like deformability or elasticity, of cells can in some cases be indicative of the health of the organism they originate from. In this work, we explore the potential of deformability and other mechanical parameters of individual red blood cells (RBCs) from humans as a marker...... but does so far not allow for subsequent investigations of single "interesting" cells. The paper is a progress report with preliminary results based on the different strategies, we have pursued....

  11. Single-cell proteomics: potential implications for cancer diagnostics.

    Science.gov (United States)

    Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

    2016-01-01

    Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

  12. Spatial reconstruction of single-cell gene expression data.

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

    2015-05-01

    Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

  13. Spatial reconstruction of single-cell gene expression

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  14. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

    Science.gov (United States)

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2016-01-12

    Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Distribution of inorganic elements in single cells of Chara corallina

    International Nuclear Information System (INIS)

    Li Zijie; Zhang Zhiyong; Chai Zhifang; Yu Ming; Zhou Yunlong

    2005-01-01

    There are actually 20 chemical elements necessary or beneficial for plant growth. Carbon, hydrogen, and oxygen are supplied by air and water. The six macronutrients, nitrogen, phosphorus, potassium., calcium, magnesium, and sulfur are required by plants in large amounts. The rest of the elements are required in trace amounts (micronutrients). Essential trace elements include boron, chlorine, copper, iron, manganese, sodium, zinc, molybdenum, and nickel. Beneficial mineral elements include silicon and cobalt. The functions of the inorganic elements closely related to their destinations in plant cells. Plant cells have unique structures, including a central vacuole, plastids, and a thick cell wall that surrounds the cell membrane. Generally, it is very difficult to determine concentrations of inorganic elements in a single plant cell. Chara corallina is a freshwater plant that inhabits temperate zone ponds and lakes. It consists of alternating nodes and internodes. Each internodal segment is a single large cell, up to 10 cm in length, and 1 mm in diameter. With this species it was possible to isolate subcellular fractions with surgical methods with minimal risk of cross contamination. In this study, concentrations of magnesium, calcium, manganese, iron, copper, zinc, and molybdenum in the cell wall, cytoplasm, and vacuole of single cells of Chara corallina were determined by inductively coupled plasma mass spectrometry (ICP-MS). The distribution characteristics of these elements in the cell components were discussed.

  16. Bridging the Timescales of Single-Cell and Population Dynamics

    Science.gov (United States)

    Jafarpour, Farshid; Wright, Charles S.; Gudjonson, Herman; Riebling, Jedidiah; Dawson, Emma; Lo, Klevin; Fiebig, Aretha; Crosson, Sean; Dinner, Aaron R.; Iyer-Biswas, Srividya

    2018-04-01

    How are granular details of stochastic growth and division of individual cells reflected in smooth deterministic growth of population numbers? We provide an integrated, multiscale perspective of microbial growth dynamics by formulating a data-validated theoretical framework that accounts for observables at both single-cell and population scales. We derive exact analytical complete time-dependent solutions to cell-age distributions and population growth rates as functionals of the underlying interdivision time distributions, for symmetric and asymmetric cell division. These results provide insights into the surprising implications of stochastic single-cell dynamics for population growth. Using our results for asymmetric division, we deduce the time to transition from the reproductively quiescent (swarmer) to the replication-competent (stalked) stage of the Caulobacter crescentus life cycle. Remarkably, population numbers can spontaneously oscillate with time. We elucidate the physics leading to these population oscillations. For C. crescentus cells, we show that a simple measurement of the population growth rate, for a given growth condition, is sufficient to characterize the condition-specific cellular unit of time and, thus, yields the mean (single-cell) growth and division timescales, fluctuations in cell division times, the cell-age distribution, and the quiescence timescale.

  17. Lessons from single-cell transcriptome analysis of oxygen-sensing cells.

    Science.gov (United States)

    Zhou, Ting; Matsunami, Hiroaki

    2018-05-01

    The advent of single-cell RNA-sequencing (RNA-Seq) technology has enabled transcriptome profiling of individual cells. Comprehensive gene expression analysis at the single-cell level has proven to be effective in characterizing the most fundamental aspects of cellular function and identity. This unbiased approach is revolutionary for small and/or heterogeneous tissues like oxygen-sensing cells in identifying key molecules. Here, we review the major methods of current single-cell RNA-Seq technology. We discuss how this technology has advanced the understanding of oxygen-sensing glomus cells in the carotid body and helped uncover novel oxygen-sensing cells and mechanisms in the mice olfactory system. We conclude by providing our perspective on future single-cell RNA-Seq research directed at oxygen-sensing cells.

  18. Reliable single cell array CGH for clinical samples.

    Directory of Open Access Journals (Sweden)

    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  19. Counting Legionella cells within single amoeba host cells

    Science.gov (United States)

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  20. Cloning of Plasmodium falciparum by single-cell sorting.

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Cloning of Plasmodium falciparum by single-cell sorting

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

  2. Single and multi-frequency impedance characterization of symmetric activated carbon single capacitor cells

    Directory of Open Access Journals (Sweden)

    Suzana Sopčić

    2018-05-01

    Full Text Available Electrochemical impedance spectroscopy (EIS technique is used for characterization of single cell symmetric capacitors having different mass loadings of activated carbon (AC. Relevant values of charge storage capacitance (CT and internal resistance (ESR were evaluated by the single frequency and multi-frequency analyses of measured impedance spectra. Curve fittings were based on the non-ideal R-C model that takes into account the parasitic inductance, contributions from electrode materials/contacts and the effects of AC porosity. Higher CT and lower ESR values were obtained not only for the cell with higher mass of AC, but also using the single vs. multi-frequency approach. Lower CT and higher values of ESR that are generally obtained using the multi-frequency method and curve fittings should be related to the not ideal capacitive response of porous AC material and too high frequency chosen in applying the single frequency analysis.

  3. RF Breakdown in Normal Conducting Single-cell Structures

    CERN Document Server

    Dolgashev, Valery A; Higo, Toshiyasu; Nantista, Christopher D; Tantawi, Sami G

    2005-01-01

    Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials an...

  4. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  5. Irradiation of single cells with individual high-LET particles

    International Nuclear Information System (INIS)

    Nelson, J.M.; Braby, L.A.

    1993-01-01

    The dose-limiting normal tissue of concern when irradiating head and neck lesions is often the vascular endothelium within the treatment field. Consequently, the response of capillary endothelial cells exposed to moderate doses of high LET particles is essential for establishing exposure limits for neutron-capture therapy. In an effort to characterize the high-LET radiation biology of cultured endothelial cells, the authors are attempting to measure cellular response to single particles. The single-particle irradiation apparatus, described below, allows them to expose individual cells to known numbers of high-LET particles and follow these cells for extended periods, in order to assess the impact of individual particles on cell growth kinetics. Preliminary cell irradiation experiments have revealed complications related to the smooth and efficient operation of the equipment; these are being resolved. Therefore, the following paragraphs deal primarily with the manner by which high LET particles deposit energy, the requirements for single-cell irradiation, construction and assembly of such apparatus, and testing of experimental procedures, rather than with the radiation biology of endothelial cells

  6. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. A photoacoustic technique to measure the properties of single cells

    Science.gov (United States)

    Strohm, Eric M.; Berndl, Elizabeth S. L.; Kolios, Michael C.

    2013-03-01

    We demonstrate a new technique to non-invasively determine the diameter and sound speed of single cells using a combined ultrasonic and photoacoustic technique. Two cell lines, B16-F1 melanoma cells and MCF7 breast cancer cells were examined using this technique. Using a 200 MHz transducer, the ultrasound backscatter from a single cell in suspension was recorded. Immediately following, the cell was irradiated with a 532 nm laser and the resulting photoacoustic wave recorded by the same transducer. The melanoma cells contain optically absorbing melanin particles, which facilitated photoacoustic wave generation. MCF7 cells have negligible optical absorption at 532 nm; the cells were permeabilized and stained with trypan blue prior to measurements. The measured ultrasound and photoacoustic power spectra were compared to theoretical equations with the cell diameter and sound speed as variables (Anderson scattering model for ultrasound, and a thermoelastic expansion model for photoacoustics). The diameter and sound speed were extracted from the models where the spectral shape matched the measured signals. However the photoacoustic spectrum for the melanoma cell did not match theory, which is likely because melanin particles are located around the cytoplasm, and not within the nucleus. Therefore a photoacoustic finite element model of a cell was developed where the central region was not used to generate a photoacoustic wave. The resulting power spectrum was in better agreement with the measured signal than the thermoelastic expansion model. The MCF7 cell diameter obtained using the spectral matching method was 17.5 μm, similar to the optical measurement of 16 μm, while the melanoma cell diameter obtained was 22 μm, similar to the optical measurement of 21 μm. The sound speed measured from the MCF7 and melanoma cell was 1573 and 1560 m/s, respectively, which is within acceptable values that have been published in literature.

  8. Single cell transcriptomics of neighboring hyphae of Aspergillus niger

    Science.gov (United States)

    2011-01-01

    Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories. PMID:21816052

  9. RoboSCell: An automated single cell arraying and analysis instrument

    KAUST Repository

    Sakaki, Kelly; Foulds, Ian G.; Liu, William; Dechev, Nikolai; Burke, Robert Douglas; Park, Edward

    2009-01-01

    Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell

  10. Bubble Jet agent release cartridge for chemical single cell stimulation.

    Science.gov (United States)

    Wangler, N; Welsche, M; Blazek, M; Blessing, M; Vervliet-Scheebaum, M; Reski, R; Müller, C; Reinecke, H; Steigert, J; Roth, G; Zengerle, R; Paust, N

    2013-02-01

    We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15 pl and V = 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.

  11. Toward single cell traction microscopy within 3D collagen matrices

    International Nuclear Information System (INIS)

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels

  12. Toward single cell traction microscopy within 3D collagen matrices

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Matthew S. [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Long, Rong [Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada T6G 2G8 (Canada); Feng, Xinzeng [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Huang, YuLing [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Hui, Chung-Yuen [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Wu, Mingming, E-mail: mw272@cornell.edu [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States)

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  13. A microfluidic galvanic cell on a single layer of paper

    Science.gov (United States)

    Purohit, Krutarth H.; Emrani, Saina; Rodriguez, Sandra; Liaw, Shi-Shen; Pham, Linda; Galvan, Vicente; Domalaon, Kryls; Gomez, Frank A.; Haan, John L.

    2016-06-01

    Paper microfluidics is used to produce single layer galvanic and hybrid cells to produce energy that could power paper-based analytical sensors. When two aqueous streams are absorbed onto paper to establish co-laminar flow, the streams stay in contact with each other with limited mixing. The interface at which mixing occurs acts as a charge-transfer region, eliminating the need for a salt bridge. We designed a Cusbnd Zn galvanic cell that powers an LED when two are placed in series. We also used more powerful redox couples (formate and silver, formate and permanganate) to produce higher power density (18 and 3.1 mW mg-1 Pd). These power densities are greater than previously reported paper microfluidic fuel cells using formate or methanol. The single layer design is much more simplified than previous reports of multi-layer galvanic cells on paper.

  14. Rotational manipulation of single cells and organisms using acoustic waves.

    Science.gov (United States)

    Ahmed, Daniel; Ozcelik, Adem; Bojanala, Nagagireesh; Nama, Nitesh; Upadhyay, Awani; Chen, Yuchao; Hanna-Rose, Wendy; Huang, Tony Jun

    2016-03-23

    The precise rotational manipulation of single cells or organisms is invaluable to many applications in biology, chemistry, physics and medicine. In this article, we describe an acoustic-based, on-chip manipulation method that can rotate single microparticles, cells and organisms. To achieve this, we trapped microbubbles within predefined sidewall microcavities inside a microchannel. In an acoustic field, trapped microbubbles were driven into oscillatory motion generating steady microvortices which were utilized to precisely rotate colloids, cells and entire organisms (that is, C. elegans). We have tested the capabilities of our method by analysing reproductive system pathologies and nervous system morphology in C. elegans. Using our device, we revealed the underlying abnormal cell fusion causing defective vulval morphology in mutant worms. Our acoustofluidic rotational manipulation (ARM) technique is an easy-to-use, compact, and biocompatible method, permitting rotation regardless of optical, magnetic or electrical properties of the sample under investigation.

  15. Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

    Science.gov (United States)

    Farzi, Bahman; Cetinkaya, Cetin

    2017-09-01

    The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the

  16. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

    Science.gov (United States)

    Wu, Jincheng; Tzanakakis, Emmanuel S.

    2014-01-01

    Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

  17. Single-cell analysis of growth and cell division of the anaerobe Desulfovibrio vulgaris Hildenborough

    Directory of Open Access Journals (Sweden)

    Anouchka eFievet

    2015-12-01

    Full Text Available Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle.In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH. This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells.

  18. A Miniature Probe for Ultrasonic Penetration of a Single Cell

    Directory of Open Access Journals (Sweden)

    Mingfei Xiao

    2009-05-01

    Full Text Available Although ultrasound cavitation must be avoided for safe diagnostic applications, the ability of ultrasound to disrupt cell membranes has taken on increasing significance as a method to facilitate drug and gene delivery. A new ultrasonic resonance driving method is introduced to penetrate rigid wall plant cells or oocytes with springy cell membranes. When a reasonable design is created, ultrasound can gather energy and increase the amplitude factor. Ultrasonic penetration enables exogenous materials to enter cells without damaging them by utilizing instant acceleration. This paper seeks to develop a miniature ultrasonic probe experiment system for cell penetration. A miniature ultrasonic probe is designed and optimized using the Precise Four Terminal Network Method and Finite Element Method (FEM and an ultrasonic generator to drive the probe is designed. The system was able to successfully puncture a single fish cell.

  19. Single cell wound generates electric current circuit and cell membrane potential variations that requires calcium influx.

    Science.gov (United States)

    Luxardi, Guillaume; Reid, Brian; Maillard, Pauline; Zhao, Min

    2014-07-24

    Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

  20. Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

    Science.gov (United States)

    Zimny, Philip; Juncker, David; Reisner, Walter

    Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

  1. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage and to alleviate pollution problems. This investigation was carried out with food wastes such as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard ...

  2. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    customary food and feed sources of protein (agriculnrre and fishery) to ocher sources like single cell protein (SCP); whose production from hydrocarbons is one ... origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, algae. protozoa, mid even bacterinphagcs generally cultivated on substrates ...

  3. Modeling single cell antibody excretion on a biosensor

    NARCIS (Netherlands)

    Stojanovic, Ivan; Baumgartner, W.; van der Velden, T.J.G.; Terstappen, Leonardus Wendelinus Mathias Marie; Schasfoort, Richardus B.M.

    2016-01-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed

  4. Direct chromosome-length haplotyping by single-cell sequencing

    NARCIS (Netherlands)

    Porubský, David; Sanders, Ashley D; van Wietmarschen, Niek; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Bevova, Marianna R; Guryev, Victor; Lansdorp, Peter Michael

    Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid

  5. Preimplantation genetic diagnosis guided by single-cell genomics

    Science.gov (United States)

    2013-01-01

    Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD. PMID:23998893

  6. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, ... Pilot plant produe1io11 of single cell proteins now take place in several centre.ii in ... animal feed but little or no information has been documented as per its ...

  7. Signatures of nonlinearity in single cell noise-induced oscillations

    NARCIS (Netherlands)

    Thomas, P.; Straube, A.V.; Timmer, J.; Fleck, C.; Grima, R.

    2013-01-01

    A class of theoretical models seeks to explain rhythmic single cell data by postulating that they are generated by intrinsic noise in biochemical systems whose deterministic models exhibit only damped oscillations. The main features of such noise-induced oscillations are quantified by the power

  8. Single-cell LEP-type cavity on measurement stand

    CERN Multimedia

    CERN PhotoLab

    1982-01-01

    A single-cell cavity, made of copper, with tapered connectors for impedance measurements. It was used as a model of LEP-type superconducting cavities, to investigate impedance and higher-order modes and operated at around 600 MHz (the LEP acceleration frequency was 352.2 MHz). See 8202500.

  9. Microbeam evolution: From single cell irradiation to preclinical studies

    DEFF Research Database (Denmark)

    Ghita, Mihaela; Fernandez-Palomo, Cristian; Fukunaga, Hisanori

    2018-01-01

    Purpose: This review follows the development of microbeam technology from the early days of single cell irradiations, to investigations of specific cellular mechanisms and to the development of new treatment modalities in vivo. A number of microbeam applications are discussed with a focus on prec...... to deliver radiotherapy using plane parallel microbeams, in Microbeam Radiotherapy (MRT)....

  10. Mutation dynamics and fitness effects followed in single cells.

    Science.gov (United States)

    Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina

    2018-03-16

    Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  11. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  12. Single-cell sequencing to quantify genomic integrity in cancer

    NARCIS (Netherlands)

    van den Bos, Hilda; Bakker, Bjorn; Spierings, Diana C J; Lansdorp, Peter M; Foijer, Floris

    The use of single-cell DNA sequencing (sc-seq) techniques for the diagnosis, prognosis and treatment of cancer is a rapidly developing field. Sc-seq research is gaining momentum by decreased sequencing costs and continuous improvements in techniques. In this review, we provide an overview of recent

  13. Magnetic domain wall conduits for single cell applications

    DEFF Research Database (Denmark)

    Donolato, Marco; Torti, A.; Kostesha, Natalie

    2011-01-01

    The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls...... walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain...... walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation....

  14. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  15. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Directory of Open Access Journals (Sweden)

    Haug Trude M

    2010-11-01

    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  16. Preparation of Single Cells for Imaging Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Berman, E S; Fortson, S L; Kulp, K S; Checchi, K D; Wu, L; Felton, J S; Wu, K J

    2007-10-24

    Characterizing chemical changes within single cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Imaging biological systems with mass spectrometry (MS) has gained popularity in recent years as a method for creating precise chemical maps of biological samples. In order to obtain high-quality mass spectral images that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell-culture components are removed from the cell surface and the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging MS that preserves the cellular contents for investigation and removes the majority of the interfering species from the extracellular matrix. Using this method, we obtain excellent imaging results and reproducibility in three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique allows routine imaging MS analysis of cultured cells, allowing for any number of experiments aimed at furthering scientific understanding of molecular processes within individual cells.

  17. Correlated receptor transport processes buffer single-cell heterogeneity.

    Directory of Open Access Journals (Sweden)

    Stefan M Kallenberger

    2017-09-01

    Full Text Available Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

  18. Advances of Single-Cell Sequencing Technique in Tumors

    Directory of Open Access Journals (Sweden)

    Ji-feng FENG

    2017-03-01

    Full Text Available With the completion of human genome project (HGP and the international HapMap project as well as rapid development of high-throughput biochip technology, whole genomic sequencing-targeted analysis of genomic structures has been primarily finished. Application of single cell for the analysis of the whole genomics is not only economical in material collection, but more importantly, the cell will be more purified, and the laboratory results will be more accurate and reliable. Therefore, exploration and analysis of hereditary information of single tumor cells has become the dream of all researchers in the field of basic research of tumors. At present, single-cell sequencing (SCS on malignancies has been widely used in the studies of pathogeneses of multiple malignancies, such as glioma, renal cancer and hematologic neoplasms, and in the studies of the metastatic mechanism of breast cancer by some researchers. This study mainly reviewed the SCS, the mechanisms and the methods of SCS in isolating tumor cells, and application of SCS technique in tumor-related basic research and clinical treatment.

  19. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  20. Capturing Three-Dimensional Genome Organization in Individual Cells by Single-Cell Hi-C.

    Science.gov (United States)

    Nagano, Takashi; Wingett, Steven W; Fraser, Peter

    2017-01-01

    Hi-C is a powerful method to investigate genome-wide, higher-order chromatin and chromosome conformations averaged from a population of cells. To expand the potential of Hi-C for single-cell analysis, we developed single-cell Hi-C. Similar to the existing "ensemble" Hi-C method, single-cell Hi-C detects proximity-dependent ligation events between cross-linked and restriction-digested chromatin fragments in cells. A major difference between the single-cell Hi-C and ensemble Hi-C protocol is that the proximity-dependent ligation is carried out in the nucleus. This allows the isolation of individual cells in which nearly the entire Hi-C procedure has been carried out, enabling the production of a Hi-C library and data from individual cells. With this new method, we studied genome conformations and found evidence for conserved topological domain organization from cell to cell, but highly variable interdomain contacts and chromosome folding genome wide. In addition, we found that the single-cell Hi-C protocol provided cleaner results with less technical noise suggesting it could be used to improve the ensemble Hi-C technique.

  1. Optimization of magnetic switches for single particle and cell transport

    Energy Technology Data Exchange (ETDEWEB)

    Abedini-Nassab, Roozbeh; Yellen, Benjamin B., E-mail: yellen@duke.edu [Department of Mechanical Engineering and Materials Science, Duke University, Box 90300 Hudson Hall, Durham, North Carolina 27708 (United States); Joint Institute, University of Michigan—Shanghai Jiao Tong University, Shanghai Jiao Tong University, Shanghai 200240 (China); Murdoch, David M. [Department of Medicine, Duke University, Durham, North Carolina 27708 (United States); Kim, CheolGi [Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873 (Korea, Republic of)

    2014-06-28

    The ability to manipulate an ensemble of single particles and cells is a key aim of lab-on-a-chip research; however, the control mechanisms must be optimized for minimal power consumption to enable future large-scale implementation. Recently, we demonstrated a matter transport platform, which uses overlaid patterns of magnetic films and metallic current lines to control magnetic particles and magnetic-nanoparticle-labeled cells; however, we have made no prior attempts to optimize the device geometry and power consumption. Here, we provide an optimization analysis of particle-switching devices based on stochastic variation in the particle's size and magnetic content. These results are immediately applicable to the design of robust, multiplexed platforms capable of transporting, sorting, and storing single cells in large arrays with low power and high efficiency.

  2. High resolution ultrasound and photoacoustic imaging of single cells

    Directory of Open Access Journals (Sweden)

    Eric M. Strohm

    2016-03-01

    Full Text Available High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

  3. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  4. High resolution imaging of surface patterns of single bacterial cells

    International Nuclear Information System (INIS)

    Greif, Dominik; Wesner, Daniel; Regtmeier, Jan; Anselmetti, Dario

    2010-01-01

    We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

  5. Advancing haematopoietic stem and progenitor cell biology through single-cell profiling

    OpenAIRE

    Hamey, Fiona; Nestorowa, Sonia; Wilson, Nicola Kaye; Göttgens, Berthold

    2016-01-01

    Haematopoietic stem and progenitor cells (HSPCs) sit at the top of the haematopoietic hierarchy, and their fate choices need to be carefully controlled to ensure balanced production of all mature blood cell types. As cell fate decisions are made at the level of the individual cells, recent technological advances in measuring gene and protein expression in increasingly large numbers of single cells have been rapidly adopted to study both normal and pathological HSPC function. In this review we...

  6. High Throughput Single-cell and Multiple-cell Micro-encapsulation

    OpenAIRE

    Lagus, Todd P.; Edd, Jon F.

    2012-01-01

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of ...

  7. Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

    Science.gov (United States)

    Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

    2016-01-01

    Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

  8. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  9. Opto-acoustic microscopy reveals adhesion mechanics of single cells

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Zc, as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZc reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, Km, that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, Sr/St. We show that Km can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while Sr/St is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  10. Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

    Science.gov (United States)

    Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

    2011-01-01

    Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

  11. Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

    Directory of Open Access Journals (Sweden)

    Jian-Feng Li

    Full Text Available Protein-protein interactions (PPIs constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

  12. Seeding of single hemopoietic stem cells and self renewal of committed stem cells

    International Nuclear Information System (INIS)

    Brecher, G.

    1986-01-01

    Single cells and two to five proliferating cells were transfused into mice whose own stem cells had been killed by irradiation. When a small inoculum of 50,000 AB marrow cells was given only 4 of 20 recipients survived, but all 4 had only PGK A enzyme in their peripheral blood cells. The results indicate that the survivors received a single pluripotential stem cell capable of proliferating. Survivors showed no deterioration in their blood picture after many months. It was concluded that there is no clonal succession in the marrow cells. Further studies with transfusions of 100,000 and 10,000,000 marrow cells after lethal irradiation suggest that there is production of committed stem cells with significant self-renewal

  13. Ciliary heterogeneity within a single cell: the Paramecium model.

    Science.gov (United States)

    Aubusson-Fleury, Anne; Cohen, Jean; Lemullois, Michel

    2015-01-01

    Paramecium is a single cell able to divide in its morphologically differentiated stage that has many cilia anchored at its cell surface. Many thousands of cilia are thus assembled in a short period of time during division to duplicate the cell pattern while the cell continues swimming. Most, but not all, of these sensory cilia are motile and involved in two main functions: prey capture and cell locomotion. These cilia display heterogeneity, both in their length and their biochemical properties. Thanks to these properties, as well as to the availability of many postgenomic tools and the possibility to follow the regrowth of cilia after deciliation, Paramecium offers a nice opportunity to study the assembly of the cilia, as well as the genesis of their diversity within a single cell. In this paper, after a brief survey of Paramecium morphology and cilia properties, we describe the tools and the protocols currently used for immunofluorescence, transmission electron microscopy, and ultrastructural immunocytochemistry to analyze cilia, with special recommendations to overcome the problem raised by cilium diversity. Copyright © 2015. Published by Elsevier Inc.

  14. Single-cell force spectroscopy of pili-mediated adhesion

    Science.gov (United States)

    Sullan, Ruby May A.; Beaussart, Audrey; Tripathi, Prachi; Derclaye, Sylvie; El-Kirat-Chatel, Sofiane; Li, James K.; Schneider, Yves-Jacques; Vanderleyden, Jos; Lebeer, Sarah; Dufrêne, Yves F.

    2013-12-01

    Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive single-cell force spectroscopy (SCFS), we quantify the adhesion forces between individual bacteria and biotic (mucin, intestinal cells) or abiotic (hydrophobic monolayers) surfaces. On hydrophobic surfaces, bacterial pili strengthen adhesion through remarkable nanospring properties, which - presumably - enable the bacteria to resist high shear forces under physiological conditions. On mucin, nanosprings are more frequent and adhesion forces larger, reflecting the influence of specific pili-mucin bonds. Interestingly, these mechanical responses are no longer observed on human intestinal Caco-2 cells. Rather, force curves exhibit constant force plateaus with extended ruptures reflecting the extraction of membrane nanotethers. These single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces (nanosprings, nanotethers), and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells (probiotics, pathogens).

  15. Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome

    Science.gov (United States)

    Teschendorff, Andrew E.; Enver, Tariq

    2017-01-01

    The ability to quantify differentiation potential of single cells is a task of critical importance. Here we demonstrate, using over 7,000 single-cell RNA-Seq profiles, that differentiation potency of a single cell can be approximated by computing the signalling promiscuity, or entropy, of a cell's transcriptome in the context of an interaction network, without the need for feature selection. We show that signalling entropy provides a more accurate and robust potency estimate than other entropy-based measures, driven in part by a subtle positive correlation between the transcriptome and connectome. Signalling entropy identifies known cell subpopulations of varying potency and drug resistant cancer stem-cell phenotypes, including those derived from circulating tumour cells. It further reveals that expression heterogeneity within single-cell populations is regulated. In summary, signalling entropy allows in silico estimation of the differentiation potency and plasticity of single cells and bulk samples, providing a means to identify normal and cancer stem-cell phenotypes. PMID:28569836

  16. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

    NARCIS (Netherlands)

    Schoeman, R.M.; Kemna, Evelien; Wolbers, F.; van den Berg, Albert

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped

  17. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  18. Cell biochemistry studied by single-molecule imaging.

    Science.gov (United States)

    Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

    2006-11-01

    Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

  19. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei

    2015-12-09

    Background In recent years, high throughput and non-invasive Raman spectrometry technique has matured as an effective approach to identification of individual cells by species, even in complex, mixed populations. Raman profiling is an appealing optical microscopic method to achieve this. To fully utilize Raman proling for single-cell analysis, an extensive understanding of Raman spectra is necessary to answer questions such as which filtering methodologies are effective for pre-processing of Raman spectra, what strains can be distinguished by Raman spectra, and what features serve best as Raman-based biomarkers for single-cells, etc. Results In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

  20. Affinity of antibody secreted by a single cell

    International Nuclear Information System (INIS)

    Doran, D.M.

    1978-01-01

    It was the intention of this research to measure the affinity of antibody secreted by a single cell, and to describe the spectrum of affinities displayed in response to antigenic stimulation. The single cell secreting specific antibody was isolated by means of the hemolytic plaque assay. The amount of antibody secreted by the cell was to be measured through the use of a solid phase radioimmunoassay. The affinity of the antibody would be estimated by comparing the diameter of the plaque, and the amount of antibody secreted, with a mathematical theory of the formation of a plaque in agar. As a test system, a solid phase radioimmunoassay was developed for human serum albumin using antibody coupled to Sephadex. A sensitivity of 1 nanogram was attained with this assay. A solid phase radioimmunoassay for mouse immunoglobulin M was developed, using antibody coupled to Sepharose. The sensitivity attained with this assay was only on the order of 10 micrograms. The mouse immunoglobulin M radioimmunoassay was not sensitive enough to measure the amount of antibody secreted by a single cell. From a theoretical equation, the relationship between antibody affinity, plaque diameter and antibody secretion rate was calculated for the experimental conditions used in this research. By assuming a constant antibody secretion rate, an effective binding constant for the antibody was estimated from the average plaque diameters. This effective binding constant was observed to increase during the immune response

  1. Abscisic Acid Induces Rapid Reductions in Mesophyll Conductance to Carbon Dioxide.

    Directory of Open Access Journals (Sweden)

    Giuseppe Sorrentino

    Full Text Available The rate of photosynthesis (A of plants exposed to water deficit is a function of stomatal (gs and mesophyll (gm conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci. Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis.

  2. Potentials of single-cell biology in identification and validation of disease biomarkers.

    Science.gov (United States)

    Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  3. Current Developments in Prokaryotic Single Cell Whole Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Goudeau, Danielle; Nath, Nandita; Ciobanu, Doina; Cheng, Jan-Fang; Malmstrom, Rex

    2014-03-14

    Our approach to prokaryotic single-cell Whole Genome Amplification at the JGI continues to evolve. To increase both the quality and number of single-cell genomes produced, we explore all aspects of the process from cell sorting to sequencing. For example, we now utilize specialized reagents, acoustic liquid handling, and reduced reaction volumes eliminate non-target DNA contamination in WGA reactions. More specifically, we use a cleaner commercial WGA kit from Qiagen that employs a UV decontamination procedure initially developed at the JGI, and we use the Labcyte Echo for tip-less liquid transfer to set up 2uL reactions. Acoustic liquid handling also dramatically reduces reagent costs. In addition, we are exploring new cell lysis methods including treatment with Proteinase K, lysozyme, and other detergents, in order to complement standard alkaline lysis and allow for more efficient disruption of a wider range of cells. Incomplete lysis represents a major hurdle for WGA on some environmental samples, especially rhizosphere, peatland, and other soils. Finding effective lysis strategies that are also compatible with WGA is challenging, and we are currently assessing the impact of various strategies on genome recovery.

  4. RF Breakdown in Normal Conducting Single-Cell Structures

    International Nuclear Information System (INIS)

    Dolgashev, V.A.; Nantista, C.D.; Tantawi, S.G.; Higashi, Y.; Higo, T.

    2006-01-01

    Operating accelerating gradient in normal conducting accelerating structures is often limited by rf breakdown. The limit depends on multiple parameters, including input rf power, rf circuit, cavity shape and material. Experimental and theoretical study of the effects of these parameters on the breakdown limit in full scale structures is difficult and costly. We use 11.4 GHz single-cell traveling wave and standing wave accelerating structures for experiments and modeling of rf breakdown behavior. These test structures are designed so that the electromagnetic fields in one cell mimic the fields in prototype multicell structures for the X-band linear collider. Fields elsewhere in the test structures are significantly lower than that of the single cell. The setup uses matched mode converters that launch the circular TM 01 mode into short test structures. The test structures are connected to the mode launchers with vacuum rf flanges. This setup allows economic testing of different cell geometries, cell materials and preparation techniques with short turn-around time. Simple 2D geometry of the test structures simplifies modeling of the breakdown currents and their thermal effects

  5. Single cell detection using a magnetic zigzag nanowire biosensor.

    Science.gov (United States)

    Huang, Hao-Ting; Ger, Tzong-Rong; Lin, Ya-Hui; Wei, Zung-Hang

    2013-08-07

    A magnetic zigzag nanowire device was designed for single cell biosensing. Nanowires with widths of 150, 300, 500, and 800 nm were fabricated on silicon trenches by electron beam lithography, electron beam evaporation, and lift-off processes. Magnetoresistance measurements were performed before and after the attachment of a single magnetic cell to the nanowires to characterize the magnetic signal change due to the influence of the magnetic cell. Magnetoresistance responses were measured in different magnetic field directions, and the results showed that this nanowire device can be used for multi-directional detection. It was observed that the highest switching field variation occurred in a 150 nm wide nanowire when the field was perpendicular to the substrate plane. On the other hand, the highest magnetoresistance ratio variation occurred in a 800 nm wide nanowire also when the field was perpendicular to the substrate plane. Besides, the trench-structured substrate proposed in this study can fix the magnetic cell to the sensor in a fluid environment, and the stray field generated by the corners of the magnetic zigzag nanowires has the function of actively attracting the magnetic cells for detection.

  6. Implementation of stimulated Raman scattering microscopy for single cell analysis

    Science.gov (United States)

    D'Arco, Annalisa; Ferrara, Maria Antonietta; Indolfi, Maurizio; Tufano, Vitaliano; Sirleto, Luigi

    2017-05-01

    In this work, we present successfully realization of a nonlinear microscope, not purchasable in commerce, based on stimulated Raman scattering. It is obtained by the integration of a femtosecond SRS spectroscopic setup with an inverted research microscope equipped with a scanning unit. Taking account of strength of vibrational contrast of SRS, it provides label-free imaging of single cell analysis. Validation tests on images of polystyrene beads are reported to demonstrate the feasibility of the approach. In order to test the microscope on biological structures, we report and discuss the label-free images of lipid droplets inside fixed adipocyte cells.

  7. Single cell analysis contemporary research and clinical applications

    CERN Document Server

    Cossarizza, Andrea

    2017-01-01

    This book highlights the current state of the art in single cell analysis, an area that involves many fields of science – from clinical hematology, functional analysis and drug screening, to platelet and microparticle analysis, marine biology and fundamental cancer research. This book brings together an eclectic group of current applications, all of which have a significant impact on our current state of knowledge. The authors of these chapters are all pioneering researchers in the field of single cell analysis. The book will not only appeal to those readers more focused on clinical applications, but also those interested in highly technical aspects of the technologies. All of the technologies identified utilize unique applications of photon detection systems.

  8. A method of combined single-cell electrophysiology and electroporation.

    Science.gov (United States)

    Graham, Lyle J; Del Abajo, Ricardo; Gener, Thomas; Fernandez, Eduardo

    2007-02-15

    This paper describes a method of extracellular recording and subsequent electroporation with the same electrode in single retinal ganglion cells in vitro. We demonstrate anatomical identification of neurons whose receptive fields were measured quantitatively. We discuss how this simple method should also be applicable for the delivery of a variety of intracellular agents, including gene delivery, to physiologically characterized neurons, both in vitro and in vivo.

  9. Identification of innate lymphoid cells in single-cell RNA-Seq data.

    Science.gov (United States)

    Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

    2017-07-01

    Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

  10. Compressive Force Spectroscopy: From Living Cells to Single Proteins.

    Science.gov (United States)

    Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

    2018-03-23

    One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

  11. Single-Walled Carbon Nanotubes in Solar Cells.

    Science.gov (United States)

    Jeon, Il; Matsuo, Yutaka; Maruyama, Shigeo

    2018-01-22

    Photovoltaics, more generally known as solar cells, are made from semiconducting materials that convert light into electricity. Solar cells have received much attention in recent years due to their promise as clean and efficient light-harvesting devices. Single-walled carbon nanotubes (SWNTs) could play a crucial role in these devices and have been the subject of much research, which continues to this day. SWNTs are known to outperform multi-walled carbon nanotubes (MWNTs) at low densities, because of the difference in their optical transmittance for the same current density, which is the most important parameter in comparing SWNTs and MWNTs. SWNT films show semiconducting features, which make SWNTs function as active or charge-transporting materials. This chapter, consisting of two sections, focuses on the use of SWNTs in solar cells. In the first section, we discuss SWNTs as a light harvester and charge transporter in the photoactive layer, which are reviewed chronologically to show the history of the research progress. In the second section, we discuss SWNTs as a transparent conductive layer outside of the photoactive layer, which is relatively more actively researched. This section introduces SWNT applications in silicon solar cells, organic solar cells, and perovskite solar cells each, from their prototypes to recent results. As we go along, the science and prospects of the application of solar cells will be discussed.

  12. Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

    Energy Technology Data Exchange (ETDEWEB)

    Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

    2014-03-14

    Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

  13. Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

    Science.gov (United States)

    Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

    2017-06-01

    Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

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

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

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

  15. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  16. Excess Diffuse Light Absorption in Upper Mesophyll Limits CO2 Drawdown and Depresses Photosynthesis.

    Science.gov (United States)

    Earles, J Mason; Théroux-Rancourt, Guillaume; Gilbert, Matthew E; McElrone, Andrew J; Brodersen, Craig R

    2017-06-01

    In agricultural and natural systems, diffuse light can enhance plant primary productivity due to deeper penetration into and greater irradiance of the entire canopy. However, for individual sun-grown leaves from three species, photosynthesis is actually less efficient under diffuse compared with direct light. Despite its potential impact on canopy-level productivity, the mechanism for this leaf-level diffuse light photosynthetic depression effect is unknown. Here, we investigate if the spatial distribution of light absorption relative to electron transport capacity in sun- and shade-grown sunflower ( Helianthus annuus ) leaves underlies its previously observed diffuse light photosynthetic depression. Using a new one-dimensional porous medium finite element gas-exchange model parameterized with light absorption profiles, we found that weaker penetration of diffuse versus direct light into the mesophyll of sun-grown sunflower leaves led to a more heterogenous saturation of electron transport capacity and lowered its CO 2 concentration drawdown capacity in the intercellular airspace and chloroplast stroma. This decoupling of light availability from photosynthetic capacity under diffuse light is sufficient to generate an 11% decline in photosynthesis in sun-grown but not shade-grown leaves, primarily because thin shade-grown leaves similarly distribute diffuse and direct light throughout the mesophyll. Finally, we illustrate how diffuse light photosynthetic depression could overcome enhancement in canopies with low light extinction coefficients and/or leaf area, pointing toward a novel direction for future research. © 2017 American Society of Plant Biologists. All Rights Reserved.

  17. Chip based single cell analysis for nanotoxicity assessment.

    Science.gov (United States)

    Shah, Pratikkumar; Kaushik, Ajeet; Zhu, Xuena; Zhang, Chengxiao; Li, Chen-Zhong

    2014-05-07

    Nanomaterials, because of their tunable properties and performances, have been utilized extensively in everyday life related consumable products and technology. On exposure, beyond the physiological range, nanomaterials cause health risks via affecting the function of organisms, genomic systems, and even the central nervous system. Thus, new analytical approaches for nanotoxicity assessment to verify the feasibility of nanomaterials for future use are in demand. The conventional analytical techniques, such as spectrophotometric assay-based techniques, usually require a lengthy and time-consuming process and often produce false positives, and often cannot be implemented at a single cell level measurement for studying cell behavior without interference from its surrounding environment. Hence, there is a demand for a precise, accurate, sensitive assessment for toxicity using single cells. Recently, due to the advantages of automation of fluids and minimization of human errors, the integration of a cell-on-a-chip (CoC) with a microfluidic system is in practice for nanotoxicity assessments. This review explains nanotoxicity and its assessment approaches with advantages/limitations and new approaches to overcome the confines of traditional techniques. Recent advances in nanotoxicity assessment using a CoC integrated with a microfluidic system are also discussed in this review, which may be of use for nanotoxicity assessment and diagnostics.

  18. Analyzing cell fate control by cytokines through continuous single cell biochemistry.

    Science.gov (United States)

    Rieger, Michael A; Schroeder, Timm

    2009-10-01

    Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time-lapse imaging and single cell tracking allowing constant long-term observation of molecules and behavior of single cells. (c) 2009 Wiley-Liss, Inc.

  19. Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

  20. Cell type discovery using single-cell transcriptomics: implications for ontological representation.

    Science.gov (United States)

    Aevermann, Brian D; Novotny, Mark; Bakken, Trygve; Miller, Jeremy A; Diehl, Alexander D; Osumi-Sutherland, David; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

    2018-05-01

    Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.

  1. Nano-imaging of single cells using STIM

    Energy Technology Data Exchange (ETDEWEB)

    Ren Minqin [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Department of Biochemistry, National University of Singapore (Singapore); Kan, J.A. van [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Bettiol, A.A. [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Daina, Lim [Department of Anatomy, National University of Singapore (Singapore); Gek, Chan Yee [Department of Anatomy, National University of Singapore (Singapore); Huat, Bay Boon [Department of Anatomy, National University of Singapore (Singapore); Whitlow, H.J. [Department of Physics, University of Jyvaskyla, P.O. Box 35 (YFL), FIN-40014 (Finland); Osipowicz, T. [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Watt, F. [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore)]. E-mail: phywattf@nus.edu.sg

    2007-07-15

    Scanning transmission ion microscopy (STIM) is a technique which utilizes the energy loss of high energy (MeV) ions passing through a sample to provide structural images. In this paper, we have successfully demonstrated STIM imaging of single cells at the nano-level using the high resolution capability of the proton beam writing facility at the Centre for Ion Beam Applications, National University of Singapore. MCF-7 breast cancer cells (American Type Culture Collection [ATCC]) were seeded on to silicon nitride windows, backed by a Hamamatsu pin diode acting as a particle detector. A reasonable contrast was obtained using 1 MeV protons and excellent contrast obtained using 1 MeV alpha particles. In a further experiment, nano-STIM was also demonstrated using cells seeded on to the pin diode directly, and high quality nano-STIM images showing the nucleus and multiple nucleoli were extracted before the detector was significantly damaged.

  2. Recent Advances in Microbial Single Cell Genomics Technology and Applications

    Science.gov (United States)

    Stepanauskas, R.

    2016-02-01

    Single cell genomics is increasingly utilized as a powerful tool to decipher the metabolic potential, evolutionary histories and in situ interactions of environmental microorganisms. This transformative technology recovers extensive information from cultivation-unbiased samples of individual, unicellular organisms. Thus, it does not require data binning into arbitrary phylogenetic or functional groups and therefore is highly compatible with agent-based modeling approaches. I will present several technological advances in this field, which significantly improve genomic data recovery from individual cells and provide direct linkages between cell's genomic and phenotypic properties. I will also demonstrate how these new technical capabilities help understanding the metabolic potential and viral infections of the "microbial dark matter" inhabiting aquatic and subsurface environments.

  3. Opto-acoustic microscopy reveals adhesion mechanics of single cells.

    Science.gov (United States)

    Abi Ghanem, Maroun; Dehoux, Thomas; Liu, Liwang; Le Saux, Guillaume; Plawinski, Laurent; Durrieu, Marie-Christine; Audoin, Bertrand

    2018-01-01

    Laser-generated GHz-ultrasonic-based technologies have shown the ability to image single cell adhesion and stiffness simultaneously. Using this new modality, we here demonstrate quantitative indicators to investigate contact mechanics and adhesion processes of the cell. We cultured human cells on a rigid substrate, and we used an inverted pulsed opto-acoustic microscope to generate acoustic pulses containing frequencies up to 100 GHz in the substrate. We map the reflection of the acoustic pulses at the cell-substrate interface to obtain images of the acoustic impedance of the cell, Z c , as well as of the stiffness of the interface, K, with 1 μm lateral resolution. Our results show that the standard deviation ΔZ c reveals differences between different cell types arising from the multiplicity of local conformations within the nucleus. From the distribution of K-values within the nuclear region, we extract a mean interfacial stiffness, K m , that quantifies the average contact force in areas of the cell displaying weak bonding. By analogy with classical contact mechanics, we also define the ratio of the real to nominal contact areas, S r /S t . We show that K m can be interpreted as a quantitative indicator of passive contact at metal-cell interfaces, while S r /S t is sensitive to active adhesive processes in the nuclear region. The ability to separate the contributions of passive and active adhesion processes should allow gaining insight into cell-substrate interactions, with important applications in tissue engineering.

  4. Whey utilization for single-cell protein production

    Energy Technology Data Exchange (ETDEWEB)

    Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

    1980-01-01

    The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

  5. Direct Visualization of De novo Lipogenesis in Single Living Cells

    Science.gov (United States)

    Li, Junjie; Cheng, Ji-Xin

    2014-10-01

    Increased de novo lipogenesis is being increasingly recognized as a hallmark of cancer. Despite recent advances in fluorescence microscopy, autoradiography and mass spectrometry, direct observation of de novo lipogenesis in living systems remains to be challenging. Here, by coupling stimulated Raman scattering (SRS) microscopy with isotope labeled glucose, we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Together, our results demonstrate a valuable tool to study dynamic lipid metabolism in cancer and other disorders.

  6. Limited angle STIM and PIXE tomography of single cells

    International Nuclear Information System (INIS)

    Andrea, T.; Rothermel, M.; Werner, R.; Butz, T.; Reinert, T.

    2010-01-01

    STIM (scanning transmission ion microscopy) tomography has been shown to be a valuable method for the three-dimensional characterization of microsamples. It has, however, rarely been employed for the study of single cells, since a free-standing sample is needed for an ordinary tomography experiment. This requirement places high demands on sample preparation techniques. In this study cells fixated on a substrate rather than free-standing were used for tomography. Since the substrate prevented a full rotation of the sample an algorithm for limited-angle tomography was devised. STIM projections covering only a limited angular range of ca. 120 o were supplemented with simulated projections generated from a back and forth iteration between real space and Radon space. The energy loss caused by the substrate was subtracted from each projection. The cells were reconstructed using filtered backprojection. The surface of the cells as well as some interior structures could be reconstructed. Following the STIM projections a lesser number of PIXE (particle induced X-ray emission) projections were taken in order to obtain information about the elemental distribution of the sample. From the PIXE projections the three-dimensional phosphorus distribution within the cell was reconstructed using limited-angle tomography. Superimposition of the STIM and PIXE tomograms revealed the location of intracellular structures. Whereas STIM tomography is sensitive to density contrast, which are greatest at the surface, PIXE tomography is sensitive to changes in elemental concentration. Hence, the combination of the two methods can be very fruitful, while the limited angle approach can compensate some of the difficulties associated with tomography of single cells, namely preparation difficulties and excessive sample damage.

  7. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    International Nuclear Information System (INIS)

    Saleem, M.; Cutler, A.J.

    1986-01-01

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of 14 C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 37 0 C. The heat shocked protoplasts incorporated 33% more 14 C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids

  8. Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

    Directory of Open Access Journals (Sweden)

    Jiangxin Wang

    Full Text Available Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett's esophagus (BE as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

  9. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

    NARCIS (Netherlands)

    Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

    2017-01-01

    Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression

  10. The impact of metabolism on aging and cell size in single yeast cells

    NARCIS (Netherlands)

    Huberts, Daphne

    2015-01-01

    The aim of this thesis was to determine how metabolism affects yeast aging in single yeast cells using a novel microfluidic device. We first review how cells are able to sense nutrients in their environment and then describe the use of the microfluidic dissection platform that greatly improves our

  11. How much territory can a single E. coli cell control?

    Directory of Open Access Journals (Sweden)

    Ziad W. El-Hajj

    2015-04-01

    Full Text Available Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. E. coli cells in particular are small rods, each 1-2 microns. However the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaloe's experiments on how E. coli establishes its size began with shifts between rich and poor media.Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensisis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems.This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK which are 2-4x longer than their nonmutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width.

  12. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

    Science.gov (United States)

    Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

    2016-06-24

    Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P

  13. Growth of single T cells and single thymocytes in a high cloning efficiency filler-cell free microculture system.

    Science.gov (United States)

    Chen, W F; Ewing, T; Scollay, R; Shortman, K

    1988-01-01

    A high cloning-efficiency microculture system is described in which single T cells, stimulated to divide by phorbol ester and calcium ionophore, grow rapidly under the influence of purified growth factors in the absence of other cells. The kinetics of clonal growth has been monitored over a five day period by phase-contrast microscopy. Mature peripheral T cells, and mature subpopulations from the thymus, responded with a cloning efficiency over 80%; they required IL-2 as a minimum but several other factors enhanced growth. Ly2+L3T4- thymocytes (mean doubling time 10.4 hr) grew more rapidly than Ly2-L3T4+ thymocytes (mean doubling time 15.2 hr). Early (Ly2-L3T4-) thymocytes responded with a cloning efficiency of 60%; their efficient growth was dependent on both IL-1 and IL-2. The typical Ly2+L3T4+ cortical thymocyte did not grow under these conditions.

  14. Automated assembling of single fuel cell units for use in a fuel cell stack

    Science.gov (United States)

    Jalba, C. K.; Muminovic, A.; Barz, C.; Nasui, V.

    2017-05-01

    The manufacturing of PEMFC stacks (POLYMER ELEKTROLYT MEMBRAN Fuel Cell) is nowadays still done by hand. Over hundreds of identical single components have to be placed accurate together for the construction of a fuel cell stack. Beside logistic problems, higher total costs and disadvantages in weight the high number of components produce a higher statistic interference because of faulty erection or material defects and summation of manufacturing tolerances. The saving of costs is about 20 - 25 %. Furthermore, the total weight of the fuel cells will be reduced because of a new sealing technology. Overall a one minute cycle time has to be aimed per cell at the manufacturing of these single components. The change of the existing sealing concept to a bonded sealing is one of the important requisites to get an automated manufacturing of single cell units. One of the important steps for an automated gluing process is the checking of the glue application by using of an image processing system. After bonding the single fuel cell the sealing and electrical function can be checked, so that only functional and high qualitative cells can get into further manufacturing processes.

  15. Signatures of nonlinearity in single cell noise-induced oscillations.

    Science.gov (United States)

    Thomas, Philipp; Straube, Arthur V; Timmer, Jens; Fleck, Christian; Grima, Ramon

    2013-10-21

    A class of theoretical models seeks to explain rhythmic single cell data by postulating that they are generated by intrinsic noise in biochemical systems whose deterministic models exhibit only damped oscillations. The main features of such noise-induced oscillations are quantified by the power spectrum which measures the dependence of the oscillatory signal's power with frequency. In this paper we derive an approximate closed-form expression for the power spectrum of any monostable biochemical system close to a Hopf bifurcation, where noise-induced oscillations are most pronounced. Unlike the commonly used linear noise approximation which is valid in the macroscopic limit of large volumes, our theory is valid over a wide range of volumes and hence affords a more suitable description of single cell noise-induced oscillations. Our theory predicts that the spectra have three universal features: (i) a dominant peak at some frequency, (ii) a smaller peak at twice the frequency of the dominant peak and (iii) a peak at zero frequency. Of these, the linear noise approximation predicts only the first feature while the remaining two stem from the combination of intrinsic noise and nonlinearity in the law of mass action. The theoretical expressions are shown to accurately match the power spectra determined from stochastic simulations of mitotic and circadian oscillators. Furthermore it is shown how recently acquired single cell rhythmic fibroblast data displays all the features predicted by our theory and that the experimental spectrum is well described by our theory but not by the conventional linear noise approximation. © 2013 Elsevier Ltd. All rights reserved.

  16. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo

    2017-12-07

    Optofluidics is a field with important applications in areas such as biotechnology, chemical synthesis and analytical chemistry. Optofluidic devices combine optical elements into microfluidic devices in ways that increase portability and sensitivity of analysis for diagnostic or screening purposes .In fact in these devices fluids give fine adaptability, mobility and accessibility to nanoscale photonic devices which otherwise could not be realized using conventional devices. This review describes several cases inwhich optical or microfluidic approaches are used to trap single cells in proximity of integrated optical sensor for being analysed.

  17. Single-cell atomic quantum memory for light

    International Nuclear Information System (INIS)

    Opatrny, Tomas

    2006-01-01

    Recent experiments demonstrating atomic quantum memory for light [B. Julsgaard et al., Nature 432, 482 (2004)] involve two macroscopic samples of atoms, each with opposite spin polarization. It is shown here that a single atomic cell is enough for the memory function if the atoms are optically pumped with suitable linearly polarized light, and quadratic Zeeman shift and/or ac Stark shift are used to manipulate rotations of the quadratures. This should enhance the performance of our quantum memory devices since less resources are needed and losses of light in crossing different media boundaries are avoided

  18. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo; Candeloro, Patrizio; Coluccio, Maria Laura; Di Fabrizio, Enzo M.

    2017-01-01

    Optofluidics is a field with important applications in areas such as biotechnology, chemical synthesis and analytical chemistry. Optofluidic devices combine optical elements into microfluidic devices in ways that increase portability and sensitivity of analysis for diagnostic or screening purposes .In fact in these devices fluids give fine adaptability, mobility and accessibility to nanoscale photonic devices which otherwise could not be realized using conventional devices. This review describes several cases inwhich optical or microfluidic approaches are used to trap single cells in proximity of integrated optical sensor for being analysed.

  19. Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq.

    Science.gov (United States)

    Reinius, Björn; Mold, Jeff E; Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

    2016-11-01

    Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8 + T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

  20. MicroBioRobots for single cell manipulation

    Science.gov (United States)

    Sakar, Mahmut Selman

    One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable microactuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a biointegrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 mum coated with a monolayer of the swarming Serratia marcescens . The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples

  1. Single-Cell Transcriptomics Bioinformatics and Computational Challenges

    Directory of Open Access Journals (Sweden)

    Lana Garmire

    2016-09-01

    Full Text Available The emerging single-cell RNA-Seq (scRNA-Seq technology holds the promise to revolutionize our understanding of diseases and associated biological processes at an unprecedented resolution. It opens the door to reveal the intercellular heterogeneity and has been employed to a variety of applications, ranging from characterizing cancer cells subpopulations to elucidating tumor resistance mechanisms. Parallel to improving experimental protocols to deal with technological issues, deriving new analytical methods to reveal the complexity in scRNA-Seq data is just as challenging. Here we review the current state-of-the-art bioinformatics tools and methods for scRNA-Seq analysis, as well as addressing some critical analytical challenges that the field faces.

  2. Claustral single cell reactions to tooth pulp stimulation in cats.

    Science.gov (United States)

    Jastreboff, P; Sikora, M; Frydrychowski, A; Słoniewski, P

    1983-01-01

    Single unit activity in the central region of the claustrum, evoked by electrical stimulation of tooth pulp or paws was studied on cats under chloralose anesthesia. The majority of cells responded in similar manner to stimulation of tooth pulp or paws, but there were cells with clear preference to a given type of stimulation. Latencies of reactions evoked by tooth pulp stimulation were significantly shorter than those for limb stimulation. In the former case latencies as short as 8 rns were observed. It is postulated that the central region of the claustrum receives a projection from the tooth pulp, and that in those cases with very short latency the projection is direct and does not involve the cerebral cortex.

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

    Science.gov (United States)

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

    2017-01-06

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

  4. Responses of single germinal-center B cells in T-cell-dependent microculture.

    Science.gov (United States)

    George, A; Cebra, J J

    1991-01-01

    B cells purified from the germinal centers (GCs) of murine Peyer's patches can be stimulated in a clonal microculture containing helper T cells and dendritic cells to divide and secrete immunoglobulin. Intraclonal isotype switching occurs, and a variety of immunoglobulin isotypes, including IgA, is secreted. Memory cells, which generate clones secreting IgA exclusively, are only rarely identified in the GC B-cell subset. Such memory cells can, however, be readily identified among unfractionated Peyer's patch B cells, and in non-GC subsets of B cells. The results suggest that the GC does not contain IgA memory cells that can be restimulated in vitro to secrete only IgA. When division of GC B cells is prevented by irradiation or aphidicholin treatment, a large subset that secretes IgA as the sole immunoglobulin isotype is seen, and the output of presumably single B cells is large enough to be scored by RIA. Both helper T cells and dendritic cells are required for the phenomenon. The data indicate that commitment to IgA secretion occurs in Peyer's patch GCs and suggest that the prolific cell division known to be supported in GCs may forestall terminal differentiation of preplasmablasts to immunoglobulin secretion.

  5. Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

    Science.gov (United States)

    Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

    2015-02-01

    Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells.

    Science.gov (United States)

    Roch, Aline; Giger, Sonja; Girotra, Mukul; Campos, Vasco; Vannini, Nicola; Naveiras, Olaia; Gobaa, Samy; Lutolf, Matthias P

    2017-08-09

    The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell-cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

  7. Asymmetrical effects of mesophyll conductance on fundamental photosynthetic parameters and their relationships estimated from leaf gas exchange measurements

    Science.gov (United States)

    Most previous analyses of leaf gas exchange measurements assumed an infinite value of mesophyll conductance (gm) and thus equaled CO2 partial pressures in the substomatal cavity and chloroplast. Yet an increasing number of studies have recognized that gm is finite and there is a drawdown of CO2 part...

  8. Stomatal conductance, mesophyll conductance, and trans piration efficiency in relation to leaf anatomy in rice and wheat genotypes under drought

    NARCIS (Netherlands)

    Ouyang, Wenjing; Struik, Paul C.; Yin, Xinyou; Yang, Jianchang

    2017-01-01

    Increasing leaf transpiration efficiency (TE) may provide leads for growing rice like dryland cereals such as wheat (Triticum aestivum). To explore avenues for improving TE in rice, variations in stomatal conductance (g s) and mesophyll conductance (g m) and their anatomical determinants were

  9. Mesophyll conductance to CO2 transport estimated by two independent methods: effect of variable CO2 concentration and abscisic acid

    Czech Academy of Sciences Publication Activity Database

    Vrábl, D.; Vašková, M.; Hronková, Marie; Flexas, J.; Šantrůček, Jiří

    2009-01-01

    Roč. 60, č. 8 (2009), s. 2315-2323 ISSN 0022-0957 R&D Projects: GA AV ČR(CZ) IAA601410505 Institutional research plan: CEZ:AV0Z50510513 Keywords : Carbon dioxide * mesophyll conductance * Helianthus annuus Subject RIV: ED - Physiology Impact factor: 4.271, year: 2009

  10. High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device.

    Science.gov (United States)

    Schoeman, Rogier M; Kemna, Evelien W M; Wolbers, Floor; van den Berg, Albert

    2014-02-01

    In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Quantum Dot Platform for Single-Cell Molecular Profiling

    Science.gov (United States)

    Zrazhevskiy, Pavel S.

    In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe

  12. Variability in mesophyll conductance between barley genotypes, and effects on transpiration efficiency and carbon isotope discrimination.

    Science.gov (United States)

    Barbour, Margaret M; Warren, Charles R; Farquhar, Graham D; Forrester, Guy; Brown, Hamish

    2010-07-01

    Leaf internal, or mesophyll, conductance to CO(2) (g(m)) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in g(m) and the effect of g(m) on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in g(m) was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest g(m) also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Delta(p)). These results suggest g(m) has unexplored potential to provide TE improvement within crop breeding programmes.

  13. Connecting single cell to collective cell behavior in a unified theoretical framework

    Science.gov (United States)

    George, Mishel; Bullo, Francesco; Campàs, Otger

    Collective cell behavior is an essential part of tissue and organ morphogenesis during embryonic development, as well as of various disease processes, such as cancer. In contrast to many in vitro studies of collective cell migration, most cases of in vivo collective cell migration involve rather small groups of cells, with large sheets of migrating cells being less common. The vast majority of theoretical descriptions of collective cell behavior focus on large numbers of cells, but fail to accurately capture the dynamics of small groups of cells. Here we introduce a low-dimensional theoretical description that successfully captures single cell migration, cell collisions, collective dynamics in small groups of cells, and force propagation during sheet expansion, all within a common theoretical framework. Our description is derived from first principles and also includes key phenomenological aspects of cell migration that control the dynamics of traction forces. Among other results, we explain the counter-intuitive observations that pairs of cells repel each other upon collision while they behave in a coordinated manner within larger clusters.

  14. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution.

    Science.gov (United States)

    Hu, Wen-Yang; Hu, Dan-Ping; Xie, Lishi; Li, Ye; Majumdar, Shyama; Nonn, Larisa; Hu, Hong; Shioda, Toshi; Prins, Gail S

    2017-08-01

    Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Isolation and functional interrogation of adult human prostate epithelial stem cells at single cell resolution

    Directory of Open Access Journals (Sweden)

    Wen-Yang Hu

    2017-08-01

    Full Text Available Using primary cultures of normal human prostate epithelial cells, we developed a novel prostasphere-based, label-retention assay that permits identification and isolation of stem cells at a single cell level. Their bona fide stem cell nature was corroborated using in vitro and in vivo regenerative assays and documentation of symmetric/asymmetric division. Robust WNT10B and KRT13 levels without E-cadherin or KRT14 staining distinguished individual stem cells from daughter progenitors in spheroids. Following FACS to isolate label-retaining stem cells from label-free progenitors, RNA-seq identified unique gene signatures for the separate populations which may serve as useful biomarkers. Knockdown of KRT13 or PRAC1 reduced sphere formation and symmetric self-renewal highlighting their role in stem cell maintenance. Pathways analysis identified ribosome biogenesis and membrane estrogen-receptor signaling enriched in stem cells with NF-ĸB signaling enriched in progenitors; activities that were biologically confirmed. Further, bioassays identified heightened autophagy flux and reduced metabolism in stem cells relative to progenitors. These approaches similarly identified stem-like cells from prostate cancer specimens and prostate, breast and colon cancer cell lines suggesting wide applicability. Together, the present studies isolate and identify unique characteristics of normal human prostate stem cells and uncover processes that maintain stem cell homeostasis in the prostate gland.

  16. New tools to study biophysical properties of single molecules and single cells

    Directory of Open Access Journals (Sweden)

    Márcio S. Rocha

    2007-03-01

    Full Text Available We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.Apresentamos uma revisão de duas novas técnicas para estudar propriedades biofísicas de moléculas únicas e células únicas. Um laser incidindo em uma objetiva de microscópio de grande abertura numérica é capaz de aprisionar pequenas partículas dielétricas na região próxima ao foco. Este aparato é chamado de pinça óptica. Esta técnica tem a grande vantagem de permitir a manipulação de um objeto individual. Usamos a pinça óptica para medir a elasticidade entrópica de uma molécula única de DNA em sua interação com o fármaco Psoralen. A pinça óptica também é usada para segurar

  17. Durum and bread wheat differ in their ability to retain potassium in leaf mesophyll: implications for salinity stress tolerance.

    Science.gov (United States)

    Wu, Honghong; Shabala, Lana; Zhou, Meixue; Shabala, Sergey

    2014-10-01

    Understanding the intrinsic mechanisms involved in the differential salinity tolerance between bread wheat and durum wheat is essential for breeding salt-tolerant varieties to cope with the global salinity issue threatening future food supply. In the past, higher salinity tolerance in bread wheat compared with durum wheat has been attributed to its better ability to exclude Na(+) from uptake. Here we show that another mechanism, namely more superior K(+) retention ability in the leaf mesophyll, also contributes to this difference. A strong positive correlation (R(2) > 0.41, P varieties. However, while the above correlation was strong in bread wheat, it was statistically insignificant in durum wheat. Consistent with these findings, a significantly higher relative leaf K(+) content was found in bread wheat than in durum wheat. In contrast to root tissues, the role of voltage-gated K(+) channels in K(+) retention in the wheat mesophyll was relatively small, and non-selective cation channels played a major role in controlling intracellular K(+) homeostasis. Moreover, a significant negative correlation between NaCl-induced mesophyll H(+) flux and mesophyll K(+) retention was found, and interpreted as a compensatory mechanism employed by sensitive varieties to regain K(+) leaked into the apoplast. It is concluded that bread wheat and durum wheat show different strategies of coping with salinity, and that targeting mechanisms conferring K(+) retention in the leaf mesophyll may be a promising way to improve the overall salinity tolerance in these species. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire.

    Science.gov (United States)

    Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H

    2013-10-03

    Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  20. Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

    Science.gov (United States)

    Jeong, Jenny; Frohberg, Nicholas J; Zhou, Enlu; Sulchek, Todd; Qiu, Peng

    2018-01-01

    Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

  1. Accurately tracking single-cell movement trajectories in microfluidic cell sorting devices.

    Directory of Open Access Journals (Sweden)

    Jenny Jeong

    Full Text Available Microfluidics are routinely used to study cellular properties, including the efficient quantification of single-cell biomechanics and label-free cell sorting based on the biomechanical properties, such as elasticity, viscosity, stiffness, and adhesion. Both quantification and sorting applications require optimal design of the microfluidic devices and mathematical modeling of the interactions between cells, fluid, and the channel of the device. As a first step toward building such a mathematical model, we collected video recordings of cells moving through a ridged microfluidic channel designed to compress and redirect cells according to cell biomechanics. We developed an efficient algorithm that automatically and accurately tracked the cell trajectories in the recordings. We tested the algorithm on recordings of cells with different stiffness, and showed the correlation between cell stiffness and the tracked trajectories. Moreover, the tracking algorithm successfully picked up subtle differences of cell motion when passing through consecutive ridges. The algorithm for accurately tracking cell trajectories paves the way for future efforts of modeling the flow, forces, and dynamics of cell properties in microfluidics applications.

  2. Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos.

    Science.gov (United States)

    Wang, Xiaochen; Gong, Yuemin; Ema, Hideo

    2016-07-22

    Adult hematopoietic stem cells (HSCs), the ideal system for regenerative research, were isolated at single cell levels decades ago, whereas studies on embryonic HSCs are much more difficult. Zhou et al identified a new pre-HSC cell surface marker, CD201, by which they isolated pre-HSCs at single cell levels for further analyses. The novel expression pattern of HSC development is revealed, including the fundamental role of mammalian targets of rapamycin (mTOR) signaling pathway in HSCs emergence, and the repopulation potential of S/G2/M phase pre-HSCs. Deeper understandings of the cellular origin and developmental regulatory network of HSCs are essential to develop new strategies of generating HSCs in vitro for clinical application.

  3. Labeling of mesenchymal stem cells for MRI with single-cell sensitivity

    Directory of Open Access Journals (Sweden)

    Ariza de Schellenberger A

    2016-04-01

    Full Text Available Angela Ariza de Schellenberger,1 Harald Kratz,1 Tracy D Farr,2,3 Norbert Löwa,4 Ralf Hauptmann,1 Susanne Wagner,1 Matthias Taupitz,1 Jörg Schnorr,1 Eyk A Schellenberger1 1Department of Radiology, 2Department of Experimental Neurology, Center for Stroke Research Berlin, Charité – Universitätsmedizin Berlin, Berlin, Germany; 3School of Life Sciences, University of Nottingham, Medical School, Nottingham, UK; 4Department of Biomagnetic Signals, Physikalisch-Technische Bundesanstalt Berlin, Berlin, Germany Abstract: Sensitive cell detection by magnetic resonance imaging (MRI is an important tool for the development of cell therapies. However, clinically approved contrast agents that allow single-cell detection are currently not available. Therefore, we compared very small iron oxide nanoparticles (VSOP and new multicore carboxymethyl dextran-coated iron oxide nanoparticles (multicore particles, MCP designed by our department for magnetic particle imaging (MPI with discontinued Resovist® regarding their suitability for detection of single mesenchymal stem cells (MSC by MRI. We achieved an average intracellular nanoparticle (NP load of >10 pg Fe per cell without the use of transfection agents. NP loading did not lead to significantly different results in proliferation, colony formation, and multilineage in vitro differentiation assays in comparison to controls. MRI allowed single-cell detection using VSOP, MCP, and Resovist® in conjunction with high-resolution T2*-weighted imaging at 7 T with postprocessing of phase images in agarose cell phantoms and in vivo after delivery of 2,000 NP-labeled MSC into mouse brains via the left carotid artery. With optimized labeling conditions, a detection rate of ~45% was achieved; however, the experiments were limited by nonhomogeneous NP loading of the MSC population. Attempts should be made to achieve better cell separation for homogeneous NP loading and to thus improve NP

  4. A probabilistic cell model in background corrected image sequences for single cell analysis

    Directory of Open Access Journals (Sweden)

    Fieguth Paul

    2010-10-01

    of localizing single cells in microwells and can be adapted for the other cell types that may not have circular shape. This method can be potentially used for single cell analysis to study the temporal dynamics of cells.

  5. Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

    Science.gov (United States)

    Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

    2017-07-12

    Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

  6. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

    OpenAIRE

    Barkla, Bronwyn J.; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte and facultative CAM plant Mesembryathemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and tr...

  7. Microencapsulation of single-cell protein from various microalgae species

    Directory of Open Access Journals (Sweden)

    Purnama Sukardi

    2015-10-01

    Full Text Available ABSTRACT The objective of the research was to evaluate nutritional values of microencapsulated diet made from single cell protein of microalgae. Complete randomized design was applied using three different types of microalgae for inclusion trials i.e. (A Nannochloropsis sp., (B Chlorella sp., and (C Spirulina sp. with five replications respectively. Microencapsulated diet was produced by a modification method based on thermal cross-linking with stable temperature. Phytoplankton was cultured in sea water for which fertilized by a modification of Walne and Guillard fertilizer. The results showed that the highest value of nutrition content was Spirulina sp. and the average composition of protein, crude lipid, carbohydrate, ash, nitrogen free extract, and water content was 34.80%, 0.30%, 18.53%, 20.09%, 26.29%, and 13.32%, respectively. Organoleptically, microcapsule showed that the color of capsule was dark green and smell fresh phytoplankton. Keywords: microcapsule, single-cell protein, thermal cross-linking, microalgae, phytoplankton  ABSTRAK Tujuan penelitian adalah mengevaluasi kandungan nutrisi pakan mikrokapsul protein sel tunggal (single cell protein yang berasal dari berbagai jenis mikroalga (fitoplankton. Rancangan percobaan yang digunakan adalah rancangan acak lengkap, dengan perlakuan inklusi mikrokapsul dari jenis fitoplankton (A Nannochloropsis sp., (B Chlorella sp., dan (C Spirulina sp., masing-masing diulang lima kali. Pembuatan mikrokapsul dilakukan dengan menggunakan modifikasi metode dasar thermal cross-linking, serta menerapkan teknik pengeringan suhu konstan. Proses pembuatan mikrokapsul protein diawali dengan kultur fitoplankton jenis Nannochloropsis sp., Chlorella sp., dan Spirulina sp. Kultur dilakukan di dalam laboratorium menggunakan media air laut dan modifikasi pupuk Walne dan Guillard. Hasil penelitian menunjukkan bahwa kandungan nutrisi tertinggi terdapat pada jenis mikrokapsul protein sel tunggal yang berasal dari

  8. Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

    Science.gov (United States)

    Ilin, Yelena; Choi, Ji Sun; Harley, Brendan A C; Kraft, Mary L

    2015-11-17

    A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that

  9. Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

    Science.gov (United States)

    Zheng, Chunhong; Zheng, Liangtao; Yoo, Jae-Kwang; Guo, Huahu; Zhang, Yuanyuan; Guo, Xinyi; Kang, Boxi; Hu, Ruozhen; Huang, Julie Y; Zhang, Qiming; Liu, Zhouzerui; Dong, Minghui; Hu, Xueda; Ouyang, Wenjun; Peng, Jirun; Zhang, Zemin

    2017-06-15

    Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 singlecells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8 + T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8 + T cells and Tregs and represses the CD8 + T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

    Science.gov (United States)

    Ricolo, Delia; Deligiannaki, Myrto; Casanova, Jordi; Araújo, Sofia J

    2016-10-24

    Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    International Nuclear Information System (INIS)

    Othon, Christina M; Ringeisen, Bradley R; Wu Xingjia; Anders, Juanita J

    2008-01-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes (∼μLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 μm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth

  12. High throughput single-cell and multiple-cell micro-encapsulation.

    Science.gov (United States)

    Lagus, Todd P; Edd, Jon F

    2012-06-15

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P(k=1) of 83.7% and a double-particle encapsulation efficiency P(k=2) of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify

  13. MICROORGANISMS: A MARVELOUS SOURCE OF SINGLE CELL PROTEINS

    Directory of Open Access Journals (Sweden)

    Agam Nangul

    2013-08-01

    Full Text Available The increasing global population living below the poverty line is driving the scientific community to search for non-conventional protein sources that can replace conventional expensive ones. Microbial proteins, or single-cell protein (SCP, represent a potential future nutrient source for human food and animal feed. These microbial proteins can be grown rapidly on substrates with minimum dependence on soil, water and climate conditions. They can be produced from algae, fungi and bacteria the chief sources of SCP. It is convenient to use microorganisms for production of SCP as they grow rapidly and have high protein content. Industrially, they can be produced from algal biomass, yeast, fungi. There are several other ways of getting SCP as well. Despite numerous advantages of SCP, they have disadvantages and toxic effects too, especially related to mycotoxins and bacterial toxins.

  14. Single-cell technologies in molecular marine studies

    KAUST Repository

    Kodzius, Rimantas

    2015-01-24

    Middle Eastern countries are experiencing a renaissance, with heavy investment in both in infrastructure and science. King Abdullah University of Science and Technology (KAUST) is a new and modern university in Saudi Arabia. At the Computational Bioscience Research Center (CBRC) we are working on exploring the Red Sea and beyond, collaborating with Japanese and other research centers. We are using the environment to collect and analyze the microorganisms present. The platform being established at CBRC allows to process samples in a pipeline. The pipeline components consist of sample collection, processing and sequencing, following the in silico analysis, determining the gene functions, identifying the organisms. The genomes of microorganisms of interest are targeted modified by genome editing technology such as CRISPR and desired properties are selected by single cell instrumentation. The final output is to identify valuable microorganisms with production of bio-energy, nutrients, the food and fine chemicals.

  15. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei; Wang, Xuetao; Gao, Xin; Ren, Lihui; Su, Xiaoquan; Bu, Dongbo; Ning, Kang

    2015-01-01

    In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

  16. Symposium on single cell analysis and genomic approaches, Experimental Biology 2017 Chicago, Illinois, April 23, 2017.

    Science.gov (United States)

    Coller, Hilary A

    2017-09-01

    Emerging technologies for the analysis of genome-wide information in single cells have the potential to transform many fields of biology, including our understanding of cell states, the response of cells to external stimuli, mosaicism, and intratumor heterogeneity. At Experimental Biology 2017 in Chicago, Physiological Genomics hosted a symposium in which five leaders in the field of single cell genomics presented their recent research. The speakers discussed emerging methodologies in single cell analysis and critical issues for the analysis of single cell data. Also discussed were applications of single cell genomics to understanding the different types of cells within an organism or tissue and the basis for cell-to-cell variability in response to stimuli. Copyright © 2017 the American Physiological Society.

  17. Single cell amperometry reveals curcuminoids modulate the release of neurotransmitters during exocytosis from PC12 cells

    Science.gov (United States)

    Li, Xianchan; Mohammadi, Amir Saeid; Ewing, Andrew G.

    2016-01-01

    We used single cell amperometry to examine whether curcumin and bisdemethoxycurcumin (BDMC), substances that are suggested to affect learning and memory, can modulate monoamine release from PC12 cells. Our results indicate both curcumin and BDMC need long-term treatment (72 h in this study) to influence exocytosis effectively. By analyzing the parameters calculated from single exocytosis events, it can be concluded that curcumin and BDMC affect exocytosis through different mechanisms. Curcumin accelerates the event dynamics with no significant change of the monoamine amount released from single exocytotic events, whereas BDMC attenuates the amount from single exocytotic event with no significant change of the event dynamics. This comparison of the effect of curcumin and BDMC on exocytosis at the single cell level brings insight into their different mechanisms, which might lead to different biological actions. The effect of curcumin and BDMC on the opening and closing of the exocytotic fusion pore were also investigated. These results might be helpful for understanding the improvement of learning and memory and the anti-depression properties of curcuminoids. PMID:28579928

  18. Differentiation of a bipotential glial progenitor cell in a single cell microculture.

    Science.gov (United States)

    Temple, S; Raff, M C

    Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.

  19. Voltage controlled nano-injection system for single-cell surgery

    Science.gov (United States)

    Seger, R. Adam; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

    2015-01-01

    Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes. PMID:22899383

  20. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.

    Science.gov (United States)

    Nagano, Takashi; Lubling, Yaniv; Yaffe, Eitan; Wingett, Steven W; Dean, Wendy; Tanay, Amos; Fraser, Peter

    2015-12-01

    Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

  1. Integrin and glycocalyx mediated contributions to cell adhesion identified by single cell force spectroscopy

    International Nuclear Information System (INIS)

    Boettiger, D; Wehrle-Haller, B

    2010-01-01

    The measurement of cell adhesion using single cell force spectroscopy methods was compared with earlier methods for measuring cell adhesion. This comparison provided a means and rationale for separating components of the measurement retract curve that were due to interactions between the substrate and the glycocalyx, and interactions that were due to cell surface integrins binding to a substrate-bound ligand. The glycocalyx adhesion was characterized by multiple jumps with dispersed jump sizes that extended from 5 to 30 μm from the origin. The integrin mediated adhesion was represented by the F max (maximum detachment force), was generally within the first 5 μm and commonly detached with a single rupture cascade. The integrin peak (F max ) increases with time and the rate of increase shows large cell to cell variability with a peak ∼ 50 nN s -1 and an average rate of increase of 75 pN s -1 . This is a measure of the rate of increase in the number of adhesive integrin-ligand bonds/cell as a function of contact time.

  2. A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization.

    Science.gov (United States)

    Dickinson, Daniel J; Schwager, Francoise; Pintard, Lionel; Gotta, Monica; Goldstein, Bob

    2017-08-21

    Regulated protein-protein interactions are critical for cell signaling, differentiation, and development. For the study of dynamic regulation of protein interactions in vivo, there is a need for techniques that can yield time-resolved information and probe multiple protein binding partners simultaneously, using small amounts of starting material. Here we describe a single-cell protein interaction assay. Single-cell lysates are generated at defined time points and analyzed using single-molecule pull-down, yielding information about dynamic protein complex regulation in vivo. We established the utility of this approach by studying PAR polarity proteins, which mediate polarization of many animal cell types. We uncovered striking regulation of PAR complex composition and stoichiometry during Caenorhabditis elegans zygote polarization, which takes place in less than 20 min. PAR complex dynamics are linked to the cell cycle by Polo-like kinase 1 and govern the movement of PAR proteins to establish polarity. Our results demonstrate an approach to study dynamic biochemical events in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. FogBank: a single cell segmentation across multiple cell lines and image modalities.

    Science.gov (United States)

    Chalfoun, Joe; Majurski, Michael; Dima, Alden; Stuelten, Christina; Peskin, Adele; Brady, Mary

    2014-12-30

    Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies. We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation. First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce. We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images. FogBank produces single cell segmentation from confluent cell

  4. Tools for Genomic and Transcriptomic Analysis of Microbes at Single-Cell Level

    Directory of Open Access Journals (Sweden)

    Zixi Chen

    2017-09-01

    Full Text Available Microbiologists traditionally study population rather than individual cells, as it is generally assumed that the status of individual cells will be similar to that observed in the population. However, the recent studies have shown that the individual behavior of each single cell could be quite different from that of the whole population, suggesting the importance of extending traditional microbiology studies to single-cell level. With recent technological advances, such as flow cytometry, next-generation sequencing (NGS, and microspectroscopy, single-cell microbiology has greatly enhanced the understanding of individuality and heterogeneity of microbes in many biological systems. Notably, the application of multiple ‘omics’ in single-cell analysis has shed light on how individual cells perceive, respond, and adapt to the environment, how heterogeneity arises under external stress and finally determines the fate of the whole population, and how microbes survive under natural conditions. As single-cell analysis involves no axenic cultivation of target microorganism, it has also been demonstrated as a valuable tool for dissecting the microbial ‘dark matter.’ In this review, current state-of-the-art tools and methods for genomic and transcriptomic analysis of microbes at single-cell level were critically summarized, including single-cell isolation methods and experimental strategies of single-cell analysis with NGS. In addition, perspectives on the future trends of technology development in the field of single-cell analysis was also presented.

  5. Single-cell RNA-Seq reveals cell heterogeneity and hierarchy within mouse mammary epithelia.

    Science.gov (United States)

    Sun, Heng; Miao, Zhengqiang; Zhang, Xin; Chan, Un In; Su, Sek Man; Guo, Sen; Wong, Chris Koon Ho; Xu, Xiaoling; Deng, Chu-Xia

    2018-04-17

    The mammary gland is very intricately and well organized into distinct tissues, including epithelia, endothelia, adipocytes, and stromal and immune cells. Many mammary gland diseases, such as breast cancer arise from abnormalities in the mammary epithelium, which is mainly composed of two distinct lineages, the basal and luminal cells. Because of the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. To this end, using single-cell RNA-Seq coupled with FACS analysis and principal component analysis, we determined gene expression profiles of mammary epithelial cells of virgin and pregnant mice. These analyses revealed a much higher heterogeneity among the mammary cells than has been previously reported and enabled cell classification into distinct subgroups according to signature gene markers present in each group. We also identified and verified a rare CDH5+ cell subpopulation within a basal cell lineage as quiescent mammary stem cells (MaSCs). Moreover, using pseudo-temporal analysis, we reconstructed the developmental trajectory of mammary epithelia and uncovered distinct changes in gene expression and in biological functions of mammary cells along the developmental process. In conclusion, our work greatly refines the resolution of the cellular hierarchy in developing mammary tissues. The discovery of CDH5+ cells as MaSCs in these tissues may have implications for our understanding of the initiation, development, and pathogenesis of mammary tumors. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency

    Directory of Open Access Journals (Sweden)

    Jamie Trott

    2013-08-01

    Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

  7. Short Peptides Enhance Single Cell Adhesion and Viability onMicroarrays

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Asphahani,Fareid; Zhang, Miqin

    2007-01-19

    Single cell patterning holds important implications forbiology, biochemistry, biotechnology, medicine, and bioinformatics. Thechallenge for single cell patterning is to produce small islands hostingonly single cells and retaining their viability for a prolonged period oftime. This study demonstrated a surface engineering approach that uses acovalently bound short peptide as a mediator to pattern cells withimproved single cell adhesion and prolonged cellular viabilityon goldpatterned SiO2 substrates. The underlying hypothesis is that celladhesion is regulated bythe type, availability, and stability ofeffective cell adhesion peptides, and thus covalently bound shortpeptides would promote cell spreading and, thus, single cell adhesion andviability. The effectiveness of this approach and the underlyingmechanism for the increased probability of single cell adhesion andprolonged cell viability by short peptides were studied by comparingcellular behavior of human umbilical cord vein endothelial cells on threemodelsurfaces whose gold electrodes were immobilized with fibronectin,physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently boundLys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and bindingproperties were characterized by reflectance Fourier transform infraredspectroscopy. Both short peptides were superior to fibronectin inproducing adhesion of only single cells, whereas the covalently boundpeptide also reduced apoptosis and necrosisof adhered cells. Controllingcell spreading by peptide binding domains to regulate apoptosis andviability represents a fundamental mechanism in cell-materialsinteraction and provides an effective strategy in engineering arrays ofsingle cells.

  8. Single Cell Dissection of Human Pancreatic Islet Dysfunction in Diabetes

    Science.gov (United States)

    2017-06-01

    of memory T cells , innate cells and the differentiation potential of naive T cells during ME/CFS; and 3) To determine the T cell and innate cell ...apoptosis and the innate immune response in human pancreatic β- cells . Diabetes 64: 3808–3817. Marselli L, Thorne J, Dahiya S, Sgroi DC, Sharma A, Bonner-Weir...interactive nature of CellView aids in cell doublet identification. In the PBMC data, ‘Subcluster-analysis’ reveals a mixture of lymphoid and myeloid

  9. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application

    Directory of Open Access Journals (Sweden)

    Amelia Ahmad Khalili

    2015-11-01

    Full Text Available Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid’s velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications.

  10. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  11. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Grasieli de Oliveira Ramos

    Full Text Available Cell migration is regulated by adhesion to the extracellular matrix (ECM through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC. We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad, plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.

  12. Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells.

    Science.gov (United States)

    Ema, Hideo; Uchinomiya, Kouki; Morita, Yohei; Suda, Toshio; Iwasa, Yoh

    2016-04-07

    The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments. Copyright © 2016. Published by Elsevier Ltd.

  13. Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Jaiswal, Devina; Rad, Armin Tahmasbi [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Nieh, Mu-Ping [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269 (United States); Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States); Claffey, Kevin P. [Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Hoshino, Kazunori, E-mail: hoshino@engr.uconn.edu [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States)

    2017-04-01

    Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 µg/ml and 0.08 µg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

  14. Mixture models for single-cell assays with applications to vaccine studies

    OpenAIRE

    Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

    2013-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers...

  15. A precise pointing nanopipette for single-cell imaging via electroosmotic injection.

    Science.gov (United States)

    Lv, Jian; Qian, Ruo-Can; Hu, Yong-Xu; Liu, Shao-Chuang; Cao, Yue; Zheng, Yong-Jie; Long, Yi-Tao

    2016-11-24

    The precise transportation of fluorescent probes to the designated location in living cells is still a challenge. Here, we present a new addition to nanopipettes as a powerful tool to deliver fluorescent molecules to a given place in a single cell by electroosmotic flow, indicating favorable potential for further application in single-cell imaging.

  16. Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

    NARCIS (Netherlands)

    Amantonico, Andrea; Oh, Joo Yeon; Sobek, Jens; Heinemann, Matthias; Zenobi, Renato

    2008-01-01

    Getting a look-in: An optimized MALDI-MS procedure has been developed to detect endogenous primary metabolites directly in the cell extract. A detection limit corresponding to metabolites from less than a single cell has been attained, opening the door to single-cell metabolomics by mass

  17. An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study.

    Science.gov (United States)

    Wong, Ieong; Atsumi, Shota; Huang, Wei-Chih; Wu, Tung-Yun; Hanai, Taizo; Lam, Miu-Ling; Tang, Ping; Yang, Jian; Liao, James C; Ho, Chih-Ming

    2010-10-21

    Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.

  18. Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

    Science.gov (United States)

    Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

    2018-05-03

    Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Single cell protein production from mandarin orange peel

    Energy Technology Data Exchange (ETDEWEB)

    Nishio, N.; Nagai, S.

    1981-01-01

    As the hydrolysis of mandarin orange peel with macerating enzyme (40/sup 0/C,24 h)produced 0.59 g g/sup -1/ reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120/sup 0/C with 0.8 N H/sub 2/SO/sub 4/), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (gg/sup -1/)were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30/sup 0/C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.

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

    Directory of Open Access Journals (Sweden)

    Artémis Llamosi

    2016-02-01

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

  1. The role of nanotechnology in single-cell detection: a review.

    Science.gov (United States)

    Wang, Changling; Zhang, Yuxiang; Xia, Mingdian; Zhu, Xingxi; Qi, Shitao; Shen, Huaqiang; Liu, Tiebing; Tang, Liming

    2014-10-01

    Biological processes in single cells, such as signal transduction, DNA duplication, and protein synthesis and trafficking, occur in subcellular compartments at nanoscale level. Achieving high spatial-temporal resolution, high sensitivity, and high specificity in single-cell detection poses a great challenge. Nanotechnology, which has been widely applied in the fields of medicine, electronics, biomaterials, and energy production, has the potential to provide solutions for single-cell detection. Here we present a review of the use of nanotechnology in single-cell detection over the past two decades. First, we review the main areas of scientific interest, including morphology, ion concentration, DNA, RNA, protein, intracellular temperature, elements, and mechanical properties. Second, four categories of application of nanotechnology to single-cell detection are described: nanomanipulation, nanodevices, nanomaterials as labels, and nano Secondary ion mass spectrometry. Finally, the prospects and future trends in single-cell detection and analysis are discussed.

  2. Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0251 TITLE: “Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA...Heterogeneity in Metabolic Disease Using Single- Cell RNA-Seq 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Linus Tzu-Yen...ABSTRACT We have developed a robust protocol to generate single cell transcriptional profiles from subcutaneous adipose tissue samples of both human

  3. cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

    Science.gov (United States)

    Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

    2017-06-01

    Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

  4. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.

    be considered. We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated...... random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding....... The strain-dependent susceptibility to bacterial colonization on conventional PLL-g-PEG illustrates how bacterial diversity challenges development of “universal” antifouling coatings, and AFM single-cell force spectroscopy was proven to be a powerful tool to provide insights into the molecular mechanisms...

  5. Central dogma at the single-molecule level in living cells.

    Science.gov (United States)

    Li, Gene-Wei; Xie, X Sunney

    2011-07-20

    Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

  6. Technical aspects and recommendations for single-cell qPCR.

    Science.gov (United States)

    Ståhlberg, Anders; Kubista, Mikael

    2018-02-01

    Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    Science.gov (United States)

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  8. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape

    NARCIS (Netherlands)

    Kamperman, Tom; Henke, Sieger; Visser, Claas Willem; Karperien, Marcel; Leijten, Jeroen

    2017-01-01

    Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is

  9. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  10. FORMING SELF-ASSEMBLED CELL ARRAYS AND MEASURING THE OXYGEN CONSUMPTION RATE OF A SINGLE LIVE CELL.

    Science.gov (United States)

    Etzkorn, James R; McQuaide, Sarah C; Anderson, Judy B; Meldrum, Deirdre R; Parviz, Babak A

    2009-06-01

    We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing "single-cell" biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

  11. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

    Directory of Open Access Journals (Sweden)

    Rong Lei

    2015-01-01

    • It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.

  12. A study on the isolation of protoplasts from mesophyll cells of Dendrobium Queen Pink

    International Nuclear Information System (INIS)

    Aqeel, R.; Zehra, M.; Kazmi, S. K.; Khan, S.

    2016-01-01

    Protoplasts were successfully isolated from one month old In vitro grown plantlets of Dendrobium cultivar Queen pink. The enzyme solution used was composed of 1 percent Cellulase Onozuka R-10, 0.5 percent Macerozyme R-10, 0.1 percent Pectinase, 0.3 M mannitol, 10 mM CaCl/sub 2/.2H/sub 2/O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Protoplast highest yield with 15.7x104 protoplasts per 1.5 gm freshly chopped leaves were obtained when digested in enzyme solution for 4 hrs on a rotary shaker with an agitation speed of 45 rpm in dark conditions. Protoplasts were filtered with 45 micro m nylon sieve and washed with 0.3 M mannitol solution supplemented with 10 mM CaCl/sub 2/.2H/sub 2/O and 10 mM MES, and purified with 0.3 M sucrose solution gradient. Purification of protoplasts on a sucrose mannitol gradient yielded clean protoplasts that were free from debris. (author)

  13. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    Science.gov (United States)

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  14. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  15. Mass sensors with mechanical traps for weighing single cells in different fluids.

    Science.gov (United States)

    Weng, Yaochung; Delgado, Francisco Feijó; Son, Sungmin; Burg, Thomas P; Wasserman, Steven C; Manalis, Scott R

    2011-12-21

    We present two methods by which single cells can be mechanically trapped and continuously monitored within the suspended microchannel resonator (SMR) mass sensor. Since the fluid surrounding the trapped cell can be quickly and completely replaced on demand, our methods are well suited for measuring changes in cell size and growth in response to drugs or other chemical stimuli. We validate our methods by measuring the density of single polystyrene beads and Saccharomyces cerevisiae yeast cells with a precision of approximately 10(-3) g cm(-3), and by monitoring the growth of single mouse lymphoblast cells before and after drug treatment.

  16. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    OpenAIRE

    Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

  17. Single guard cell recordings in intact plants : light-induced hyperpolarization of the plasma membrane

    NARCIS (Netherlands)

    Roelfsema, MRG; Steinmeyer, R; Staal, M; Hedrich, R

    Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit

  18. Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

    Directory of Open Access Journals (Sweden)

    Manabu Watanabe

    2014-09-01

    Full Text Available Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line were used as a model. Single-cell capture was performed using laser capture microdissection (LCM with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells were subjected to whole genome amplification (WGA. For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100× were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100× were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

  19. Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells.

    Science.gov (United States)

    Browning, Lauren M; Lee, Kerry J; Cherukuri, Pavan K; Huang, Tao; Songkiatisak, Preeyaporn; Warren, Seth; Xu, Xiao-Hong Nancy

    2018-03-26

    ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding

  20. QSpec: online control and data analysis system for single-cell Raman spectroscopy

    Directory of Open Access Journals (Sweden)

    Lihui Ren

    2014-06-01

    Full Text Available Single-cell phenotyping is critical to the success of biological reductionism. Raman-activated cell sorting (RACS has shown promise in resolving the dynamics of living cells at the individual level and to uncover population heterogeneities in comparison to established approaches such as fluorescence-activated cell sorting (FACS. Given that the number of single-cells would be massive in any experiment, the power of Raman profiling technique for single-cell analysis would be fully utilized only when coupled with a high-throughput and intelligent process control and data analysis system. In this work, we established QSpec, an automatic system that supports high-throughput Raman-based single-cell phenotyping. Additionally, a single-cell Raman profile database has been established upon which data-mining could be applied to discover the heterogeneity among single-cells under different conditions. To test the effectiveness of this control and data analysis system, a sub-system was also developed to simulate the phenotypes of single-cells as well as the device features.

  1. Quantum cascade laser infrared spectroscopy of single cancer cells

    KAUST Repository

    Patel, Imran

    2017-03-27

    Quantum cascade laser infrared spectroscopy is a next generation novel imaging technique allowing high resolution spectral imaging of cells. We show after spectral pre-processing, identification of different cancer cell populations within minutes.

  2. Quantum cascade laser infrared spectroscopy of single cancer cells

    KAUST Repository

    Patel, Imran; Rajamanickam, Vijayakumar Palanisamy; Bertoncini, Andrea; Pagliari, Francesca; Tirinato, Luca; Laptenok, Sergey P.; Liberale, Carlo

    2017-01-01

    Quantum cascade laser infrared spectroscopy is a next generation novel imaging technique allowing high resolution spectral imaging of cells. We show after spectral pre-processing, identification of different cancer cell populations within minutes.

  3. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation

    Directory of Open Access Journals (Sweden)

    Hisham Mohammed

    2017-08-01

    Full Text Available The mouse inner cell mass (ICM segregates into the epiblast and primitive endoderm (PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.

  4. Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

    2011-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  5. The single-cell transcriptional landscape of hematopoiesis

    NARCIS (Netherlands)

    Baron, Chloé Sophie

    2018-01-01

    Hematopoietic stem cells (HSCs) are responsible for the production of all mature hematopoietic cells during the entire life of an organism. It is now known that HSCs are generated from a specific subset of endothelial cells in the main arteries of the developing mouse and zebrafish embryos. In the

  6. Genome wide single cell analysis of chemotherapy resistant metastatic cells in a case of gastroesophageal adenocarcinoma

    International Nuclear Information System (INIS)

    Hjortland, Geir Olav; Fodstad, Oystein; Smeland, Sigbjorn; Hovig, Eivind; Meza-Zepeda, Leonardo A; Beiske, Klaus; Ree, Anne H; Tveito, Siri; Hoifodt, Hanne; Bohler, Per J; Hole, Knut H; Myklebost, Ola

    2011-01-01

    Metastatic progression due to development or enrichment of therapy-resistant tumor cells is eventually lethal. Molecular characterization of such chemotherapy resistant tumor cell clones may identify markers responsible for malignant progression and potential targets for new treatment. Here, in a case of stage IV adenocarcinoma of the gastroesophageal junction, we report the successful genome wide analysis using array comparative genomic hybridization (CGH) of DNA from only fourteen tumor cells using a bead-based single cell selection method from a bone metastasis progressing during chemotherapy. In a case of metastatic adenocarcinoma of the gastroesophageal junction, the progression of bone metastasis was observed during a chemotherapy regimen of epirubicin, oxaliplatin and capecitabine, whereas lung-, liver and lymph node metastases as well as the primary tumor were regressing. A bone marrow aspirate sampled at the site of progressing metastasis in the right iliac bone was performed, and single cell molecular analysis using array-CGH of Epithelial Specific Antigen (ESA)-positive metastatic cells, and revealed two distinct regions of amplification, 12p12.1 and 17q12-q21.2 amplicons, containing the KRAS (12p) and ERBB2 (HER2/NEU) (17q) oncogenes. Further intrapatient tumor heterogeneity of these highlighted gene copy number changes was analyzed by fluorescence in situ hybridization (FISH) in all available primary and metastatic tumor biopsies, and ErbB2 protein expression was investigated by immunohistochemistry. ERBB2 was heterogeneously amplified by FISH analysis in the primary tumor, as well as liver and bone metastasis, but homogenously amplified in biopsy specimens from a progressing bone metastasis after three initial cycles of chemotherapy, indicating a possible enrichment of erbB2 positive tumor cells in the progressing bone marrow metastasis during chemotherapy. A similar amplification profile was detected for wild-type KRAS, although more heterogeneously

  7. RoboSCell: An automated single cell arraying and analysis instrument

    KAUST Repository

    Sakaki, Kelly

    2009-09-09

    Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument\\'s capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester-all in a fully automated fashion. © 2009 Springer Science+Business Media, LLC.

  8. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

  9. Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

    DEFF Research Database (Denmark)

    Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori

    2017-01-01

    , an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood...... a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice....

  10. Fluidic Logic Used in a Systems Approach to Enable Integrated Single-cell Functional Analysis

    Directory of Open Access Journals (Sweden)

    Naveen Ramalingam

    2016-09-01

    Full Text Available The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide-spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity. Currently, it is difficult to perturb single cells in a controlled environment, monitor and measure the response due to perturbation, and link these response measurements to downstream genomic and transcriptomic analysis. In order to address this challenge, we developed a platform to integrate and miniaturize many of the experimental steps required to study single-cell function. The heart of this platform is an elastomer-based Integrated Fluidic Circuit (IFC that uses fluidic logic to select and sequester specific single cells based on a phenotypic trait for downstream experimentation. Experiments with sequestered cells that have been performed include on-chip culture, exposure to a variety of stimulants, and post-exposure image-based response analysis, followed by preparation of the mRNA transcriptome for massively parallel sequencing analysis. The flexible system embodies experimental design and execution that enable routine functional studies of single cells.

  11. Performance of planar single cell lanthanum gallate based solid oxide fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Maffei, N.; Kuriakose, A.K. [Materials Technology Labs., CANMET, Natural Resources Canada, Ottawa, ON (Canada)

    1998-09-01

    A novel synthesis of high purity, single phase strontium-magnesium doped lanthanum gallate through a nitrate route is described. The prepared powder is formed into planar monolithic elements by uniaxial pressing followed by isostatic pressing and sintering. XRD analysis of the sintered elements reveal no detectable secondary phases. The performance of the electrolyte in solid oxide fuel cells (SOFC) with three different anode/cathode combinations tested at 700 C with respect to the J-V and power density is reported. The data show that the characteristics of this SOFC are strongly dependent on the particular anode/cathode system chosen. (orig.)

  12. Performance of planar single cell lanthanum gallate based solid oxide fuel cells

    Science.gov (United States)

    Maffei, N.; Kuriakose, A. K.

    A novel synthesis of high purity, single phase strontium-magnesium doped lanthanum gallate through a nitrate route is described. The prepared powder is formed into planar monolithic elements by uniaxial pressing followed by isostatic pressing and sintering. XRD analysis of the sintered elements reveal no detectable secondary phases. The performance of the electrolyte in solid oxide fuel cells (SOFC) with three different anode/cathode combinations tested at 700°C with respect to the J- V and power density is reported. The data show that the characteristics of this SOFC are strongly dependent on the particular anode/cathode system chosen.

  13. Using micro-patterned sensors and cell self-assembly for measuring the oxygen consumption rate of single cells

    International Nuclear Information System (INIS)

    Etzkorn, James R; Parviz, Babak A; Wu, Wen-Chung; Tian, Zhiyuan; Kim, Prince; Jang, Sei-Hum; Jen, Alex K-Y; Meldrum, Deirdre R

    2010-01-01

    We present a method for self-assembling arrays of live single cells on a glass chip using a photopatternable polymer to form micro-traps. We have studied the single-cell self-assembly method and optimized the process to obtain a 52% yield of single-trapped cells. We also report a method to measure the oxygen consumption rate of a single cell using micro-patterned sensors. These molecular oxygen sensors were fabricated around each micro-trap allowing optical interrogation of oxygen concentration in the immediate environment of the trapped cell. Micromachined micro-wells were then used to seal the trap, sensor and cell in order to determine the oxygen consumption rate of single cells. These techniques reported here add to the collection of tools for performing 'singe-cell' biology. An oxygen consumption rate of 1.05 ± 0.28 fmol min −1 was found for a data set consisting of 25 single A549 cells.

  14. Unravelling biology and shifting paradigms in cancer with single-cell sequencing.

    Science.gov (United States)

    Baslan, Timour; Hicks, James

    2017-08-24

    The fundamental operative unit of a cancer is the genetically and epigenetically innovative single cell. Whether proliferating or quiescent, in the primary tumour mass or disseminated elsewhere, single cells govern the parameters that dictate all facets of the biology of cancer. Thus, single-cell analyses provide the ultimate level of resolution in our quest for a fundamental understanding of this disease. Historically, this quest has been hampered by technological shortcomings. In this Opinion article, we argue that the rapidly evolving field of single-cell sequencing has unshackled the cancer research community of these shortcomings. From furthering an elemental understanding of intra-tumoural genetic heterogeneity and cancer genome evolution to illuminating the governing principles of disease relapse and metastasis, we posit that single-cell sequencing promises to unravel the biology of all facets of this disease.

  15. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  16. Study of a Microfluidic Chip Integrating Single Cell Trap and 3D Stable Rotation Manipulation

    Directory of Open Access Journals (Sweden)

    Liang Huang

    2016-08-01

    Full Text Available Single cell manipulation technology has been widely applied in biological fields, such as cell injection/enucleation, cell physiological measurement, and cell imaging. Recently, a biochip platform with a novel configuration of electrodes for cell 3D rotation has been successfully developed by generating rotating electric fields. However, the rotation platform still has two major shortcomings that need to be improved. The primary problem is that there is no on-chip module to facilitate the placement of a single cell into the rotation chamber, which causes very low efficiency in experiment to manually pipette single 10-micron-scale cells into rotation position. Secondly, the cell in the chamber may suffer from unstable rotation, which includes gravity-induced sinking down to the chamber bottom or electric-force-induced on-plane movement. To solve the two problems, in this paper we propose a new microfluidic chip with manipulation capabilities of single cell trap and single cell 3D stable rotation, both on one chip. The new microfluidic chip consists of two parts. The top capture part is based on the least flow resistance principle and is used to capture a single cell and to transport it to the rotation chamber. The bottom rotation part is based on dielectrophoresis (DEP and is used to 3D rotate the single cell in the rotation chamber with enhanced stability. The two parts are aligned and bonded together to form closed channels for microfluidic handling. Using COMSOL simulation and preliminary experiments, we have verified, in principle, the concept of on-chip single cell traps and 3D stable rotation, and identified key parameters for chip structures, microfluidic handling, and electrode configurations. The work has laid a solid foundation for on-going chip fabrication and experiment validation.

  17. Decoding Signal Processing at the Single-Cell Level

    Energy Technology Data Exchange (ETDEWEB)

    Wiley, H. Steven

    2017-12-01

    The ability of cells to detect and decode information about their extracellular environment is critical to generating an appropriate response. In multicellular organisms, cells must decode dozens of signals from their neighbors and extracellular matrix to maintain tissue homeostasis while still responding to environmental stressors. How cells detect and process information from their surroundings through a surprisingly limited number of signal transduction pathways is one of the most important question in biology. Despite many decades of research, many of the fundamental principles that underlie cell signal processing remain obscure. However, in this issue of Cell Systems, Gillies et al present compelling evidence that the early response gene circuit can act as a linear signal integrator, thus providing significant insight into how cells handle fluctuating signals and noise in their environment.

  18. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Science.gov (United States)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  19. Allogeneic cell therapy bioprocess economics and optimization: single-use cell expansion technologies.

    Science.gov (United States)

    Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

    2014-01-01

    For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 10(9) cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. © 2013 Wiley Periodicals, Inc.

  20. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells

    International Nuclear Information System (INIS)

    Yamada, Yoji; Sakurada, Kazuhiro; Takeda, Yukiji; Gojo, Satoshi; Umezawa, Akihiro

    2007-01-01

    Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo

  1. Single cell low dose studies of bystander cell killing with targeted ultrasoft x-rays

    International Nuclear Information System (INIS)

    Schettino, G.; Prise, K.M.; Folkard, M.; Vojnovic, B.; Michael, B.D.; Wu, L.; Held, K.D.

    2003-01-01

    Full text: Bystander responses have attracted considerable interest in the recent years and several investigations have reported a binary behavior with the effect triggered by very small doses and immediately reaching a plateau. The Ultrasoft X-ray Microprobe in operation at the GCI is a facility designed to precisely assess the biological response of individual cells in vitro irradiated with a sub-micron size X-ray beam . Although recent improvements have upgrade the facility with AlK and TiK X-rays, most of the bystander studies have been performed using CK X-rays of 278 eV. The high sensitivity and the accurate irradiation and revisiting of the individual samples allowed us to investigate specific characteristics of the bystander phenomenon. In particular, evidences of a dose dependency of cell killing by bystander effect have been found at doses below 0.2 Gy where no differences is observed between all cell and single cell irradiation. Recent improvements have also allowed us to individually identify the phase of the cell cycle of all samples exposed. Although the G2-S phase have been found the most sensitive in responding to the bystander signal (a factor of 1.3), cells in the G1 phase also respond significantly while the phase of the irradiated cell doesn't seem to play a critical role. The time scale of the bystander effect has also been investigated by irradiating the same sample(s) twice with a few hours gap between exposures. Results indicate that the bystander signal is transmitted within a few minutes from the irradiation as replacing the medium immediately after irradiation does not influence the response, and it doesn't depend on the number of cells irradiated (up to 5). However, after a resting time of a few hours (3 h), the system seems to reset itself and a second irradiation has been shown to trigger a further bystander effect. Finally, by considering the total amount of energy deposited in to the sample population as critical parameter (instead

  2. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    OpenAIRE

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up...

  3. Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

    Science.gov (United States)

    Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

    2011-11-01

    Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

  4. Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2017-10-01

    Full Text Available Summary: Glioblastoma (GBM is the most common primary brain cancer in adults and is notoriously difficult to treat because of its diffuse nature. We performed single-cell RNA sequencing (RNA-seq on 3,589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumor’s genome and transcriptome. We were also able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of information about tumor formation and migration. : Darmanis et al. perform single-cell transcriptomic analyses of neoplastic and stromal cells within and proximal to primary glioblastomas. The authors describe a population of neoplastic-infiltrating glioblastoma cells as well as a putative role of tumor-infiltrating immune cells in supporting tumor growth. Keywords: single cell, RNA-seq, glioma, glioblastoma, GBM, brain, heterogeneity, infiltrating, diffuse, checkpoint

  5. Single crystalline silicon solar cells with rib structure

    Directory of Open Access Journals (Sweden)

    Shuhei Yoshiba

    2017-02-01

    Full Text Available To improve the conversion efficiency of Si solar cells, we have developed a thin Si wafer-based solar cell that uses a rib structure. The open-circuit voltage of a solar cell is known to increase with deceasing wafer thickness if the cell is adequately passivated. However, it is not easy to handle very thin wafers because they are brittle and are subject to warpage. We fabricated a lattice-shaped rib structure on the rear side of a thin Si wafer to improve the wafer’s strength. A silicon nitride film was deposited on the Si wafer surface and patterned to form a mask to fabricate the lattice-shaped rib, and the wafer was then etched using KOH to reduce the thickness of the active area, except for the rib region. Using this structure in a Si heterojunction cell, we demonstrated that a high open-circuit voltage (VOC could be obtained by thinning the wafer without sacrificing its strength. A wafer with thickness of 30 μm was prepared easily using this structure. We then fabricated Si heterojunction solar cells using these rib wafers, and measured their implied VOC as a function of wafer thickness. The measured values were compared with device simulation results, and we found that the measured VOC agrees well with the simulated results. To optimize the rib and cell design, we also performed device simulations using various wafer thicknesses and rib dimensions.

  6. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Directory of Open Access Journals (Sweden)

    Bronwyn Jane Barkla

    2015-06-01

    Full Text Available One of the remarkable adaptive features of the halophyte and facultative CAM plant Mesembryathemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples was used to identify 352 significantly differing metabolites (268 after correction for FDR. Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na and Cl ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggest large alterations in Mesembryanthemum crystallinum epidermal bladder cells.

  7. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na(+) and Cl(-) ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells.

  8. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na+ and Cl− ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells. PMID:26113856

  9. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize...... of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were...

  10. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    NARCIS (Netherlands)

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical

  11. Potential in a single cancer cell to produce heterogeneous morphology, radiosensitivity and gene expression

    International Nuclear Information System (INIS)

    Ban, Sadayuki; Ishikawa, Ken-ichi; Kawai, Seiko; Koyama-Saegusa, Kumiko; Ishikawa, Atsuko; Imai, Takashi; Shimada, Yutaka; Inazawa, Johji

    2005-01-01

    Morphologically heterogeneous colonies were formed from a cultured cell line (KYSE70) established from one human esophageal carcinoma tissue. Two subclones were separated from a single clone (clone 13) of KYSE70 cells. One subclone (clone 13-3G) formed mainly mounding colonies and the other (clone 13-6G) formed flat, diffusive colonies. X-irradiation stimulated the cells to dedifferentiate from the mounding state to the flat, diffusive state. Clone 13-6G cells were more radiosensitive than the other 3 cell lines. Clustering analysis for gene expression level by oligonucleotide microarray demonstrated that in the radiosensitive clone 13-6G cells, expression of genes involved in cell adhesion was upregulated, but genes involved in the response to DNA damage stimulus were downregulated. The data demonstrated that a single cancer cell had the potential to produce progeny heterogeneous in terms of morphology, radiation sensitivity and gene expression, and irradiation enhanced the dedifferentiation of cancer cells. (author)

  12. Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

    2013-10-01

    Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

  13. A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2016-09-01

    Full Text Available Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231 are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, cyclin-dependent kinase inhibitor 1A (CDKN1A, and aurora kinase A (AURKA genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.

  14. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    John J Vincent

    Full Text Available The cell intrinsic programming that regulates mammalian primordial germ cell (PGC development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs from mouse embryonic stem cells (ESCs. Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

  15. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  16. Application of the comet assay in studies of programmed cell death (PCD in plants

    Directory of Open Access Journals (Sweden)

    Maria Charzyńska

    2014-01-01

    Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

  17. Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

    Science.gov (United States)

    Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

    2017-07-01

    In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

  18. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

    Science.gov (United States)

    Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

    2015-01-01

    We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

  19. Single cell analysis: the new frontier in 'Omics'

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Bodovitz, Steven

    2010-01-14

    Cellular heterogeneity arising from stochastic expression of genes, proteins, and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of 'Omics' technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics, and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.

  20. Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

    Science.gov (United States)

    Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

    2018-05-01

    Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

  1. Volatility of Mutator Phenotypes at Single Cell Resolution.

    Directory of Open Access Journals (Sweden)

    Scott R Kennedy

    2015-04-01

    Full Text Available Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

  2. Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

    Science.gov (United States)

    Kunicki, Matthew A; Amaya Hernandez, Laura C; Davis, Kara L; Bacchetta, Rosa; Roncarolo, Maria-Grazia

    2018-01-01

    Human CD3 + CD4 + Th cells, FOXP3 + T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3 + CD4 + T cell compartment remains questionable. In this study, we examined CD3 + CD4 + T cell populations by single-cell mass cytometry. We characterize the CD3 + CD4 + Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3 + CD4 + Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4) + FOXP3 + Treg and CD183 (CXCR3) + T-bet + Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3 + CD4 + T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3 + CD4 + T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies. Copyright © 2017 by The American Association of Immunologists, Inc.

  3. Dynamic single-cell NAD(P)H measurement reveals oscillatory metabolism throughout the E. coli cell division cycle.

    Science.gov (United States)

    Zhang, Zheng; Milias-Argeitis, Andreas; Heinemann, Matthias

    2018-02-01

    Recent work has shown that metabolism between individual bacterial cells in an otherwise isogenetic population can be different. To investigate such heterogeneity, experimental methods to zoom into the metabolism of individual cells are required. To this end, the autofluoresence of the redox cofactors NADH and NADPH offers great potential for single-cell dynamic NAD(P)H measurements. However, NAD(P)H excitation requires UV light, which can cause cell damage. In this work, we developed a method for time-lapse NAD(P)H imaging in single E. coli cells. Our method combines a setup with reduced background emission, UV-enhanced microscopy equipment and optimized exposure settings, overall generating acceptable NAD(P)H signals from single cells, with minimal negative effect on cell growth. Through different experiments, in which we perturb E. coli's redox metabolism, we demonstrated that the acquired fluorescence signal indeed corresponds to NAD(P)H. Using this new method, for the first time, we report that intracellular NAD(P)H levels oscillate along the bacterial cell division cycle. The developed method for dynamic measurement of NAD(P)H in single bacterial cells will be an important tool to zoom into metabolism of individual cells.

  4. In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

    Science.gov (United States)

    Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

    2014-10-01

    The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

  5. A single-cell scraper based on an atomic force microscope for detaching a living cell from a substrate

    Energy Technology Data Exchange (ETDEWEB)

    Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan); Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011 (Japan); Adachi, Makoto; Hashimoto, Shigetaka [Department of Mechanical Engineering, Faculty of Engineering, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8561 (Japan)

    2015-10-07

    We describe an atomic force microscope (AFM) manipulator that can detach a single, living adhesion cell from its substrate without compromising the cell's viability. The micrometer-scale cell scraper designed for this purpose was fabricated from an AFM micro cantilever using focused ion beam milling. The homemade AFM equipped with the scraper was compact and standalone and could be mounted on a sample stage of an inverted optical microscope. It was possible to move the scraper using selectable modes of operation, either a manual mode with a haptic device or a computer-controlled mode. The viability of the scraped single cells was evaluated using a fluorescence dye of calcein-acetoxymethl ester. Single cells detached from the substrate were collected by aspiration into a micropipette capillary glass using an electro-osmotic pump. As a demonstration, single HeLa cells were selectively detached from the substrate and collected by the micropipette. It was possible to recultivate HeLa cells from the single cells collected using the system.

  6. Effects of sample treatments on genome recovery via single-cell genomics

    Energy Technology Data Exchange (ETDEWEB)

    Clingenpeel, Scott [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Schwientek, Patrick [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Hugenholtz, Philip [Univ. of Queensland, Brisbane (Australia); Woyke, Tanja [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)

    2014-06-13

    It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

  7. Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

    Science.gov (United States)

    Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

    2018-03-01

    EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

  8. Single-Molecule Light-Sheet Imaging of Suspended T Cells.

    Science.gov (United States)

    Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

    2018-05-08

    Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

  9. SCNS: a graphical tool for reconstructing executable regulatory networks from single-cell genomic data.

    Science.gov (United States)

    Woodhouse, Steven; Piterman, Nir; Wintersteiger, Christoph M; Göttgens, Berthold; Fisher, Jasmin

    2018-05-25

    Reconstruction of executable mechanistic models from single-cell gene expression data represents a powerful approach to understanding developmental and disease processes. New ambitious efforts like the Human Cell Atlas will soon lead to an explosion of data with potential for uncovering and understanding the regulatory networks which underlie the behaviour of all human cells. In order to take advantage of this data, however, there is a need for general-purpose, user-friendly and efficient computational tools that can be readily used by biologists who do not have specialist computer science knowledge. The Single Cell Network Synthesis toolkit (SCNS) is a general-purpose computational tool for the reconstruction and analysis of executable models from single-cell gene expression data. Through a graphical user interface, SCNS takes single-cell qPCR or RNA-sequencing data taken across a time course, and searches for logical rules that drive transitions from early cell states towards late cell states. Because the resulting reconstructed models are executable, they can be used to make predictions about the effect of specific gene perturbations on the generation of specific lineages. SCNS should be of broad interest to the growing number of researchers working in single-cell genomics and will help further facilitate the generation of valuable mechanistic insights into developmental, homeostatic and disease processes.

  10. From single cells to tissues: interactions between the matrix and human breast cells in real time.

    Directory of Open Access Journals (Sweden)

    Clifford Barnes

    Full Text Available Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

  11. Single-cell and subcellular pharmacokinetic imaging allows insight into drug action in vivo.

    Science.gov (United States)

    Thurber, Greg M; Yang, Katy S; Reiner, Thomas; Kohler, Rainer H; Sorger, Peter; Mitchison, Tim; Weissleder, Ralph

    2013-01-01

    Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.

  12. Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

    Science.gov (United States)

    Matsumura, Taku; Tatsumi, Kazuya; Noda, Yuichiro; Nakanishi, Naoyuki; Okonogi, Atsuhito; Hirano, Kunio; Li, Liu; Osumi, Takashi; Tada, Takashi; Kotera, Hidetoshi

    2014-10-10

    The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Optical detection of singlet oxygen from single cells

    DEFF Research Database (Denmark)

    Snyder, John; Skovsen, Esben; Lambert, John D. C.

    2006-01-01

    The lowest excited electronic state of molecular oxygen, singlet molecular oxygen, O2(a 1g), is a reactive species involved in many chemical and biological processes. To better understand the roles played by singlet oxygen in biological systems, particularly at the sub-cellular level, optical tools...... including across the cell membrane into the extracellular environment. On one hand, these results demonstrate that the behavior of singlet oxygen in an intact cell can be significantly different from that inferred from model bulk studies. More generally, these results provide a new perspective...

  14. Mesophyll conductance in Zea mays responds transiently to CO2 availability: implications for transpiration efficiency in C4 crops.

    Science.gov (United States)

    Kolbe, Allison R; Cousins, Asaph B

    2018-03-01

    Mesophyll conductance (g m ) describes the movement of CO 2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C 3 -g m , little is currently known about the intraspecific variation in C 4 -g m or its responsiveness to environmental stimuli. To address these questions, g m was measured on five maize (Zea mays) lines in response to CO 2 , employing three different estimates of g m . Each of the methods indicated a significant response of g m to CO 2 . Estimates of g m were similar between methods at ambient and higher CO 2 , but diverged significantly at low partial pressures of CO 2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO 2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of g m and leaf anatomical traits. These results provide strong support for a CO 2 response of g m in Z. mays, and indicate that g m in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO 2 response of g m indicates a potential role for facilitated diffusion in C 4 -g m . These results also suggest that water-use efficiency could be enhanced in C 4 species by targeting g m . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  15. Photosynthetic response of an alpine plant, Rhododendron delavayi Franch, to water stress and recovery: the role of mesophyll conductance

    Directory of Open Access Journals (Sweden)

    Yanfei eCai

    2015-12-01

    Full Text Available Rhododendron delavayi Franch is an evergreen shrub or small tree with large scarlet flowers that makes it highly attractive as an ornamental species. The species is native to southwest China and southeast Asia, especially the Himalayan region, showing good adaptability and tolerance to drought. To understand the water stress coping mechanisms of R. delavayi, we analysed the plant’s photosynthetic performance during water stress and recovery. In particular, we looked at the regulation of stomatal (gs and mesophyll conductance (gm, and maximum rate of carboxylation (Vcmax. After four days of water stress treatment, the net CO2 assimilation rate (AN declined slightly while gs and gm were not affected and stomatal limitation (SL was therefore negligible. At this stage mesophyll conductance limitation (MCL and biochemical limitation (BL constituted the main limitation factors. After eight days of water stress treatment, AN, gs and gm had decreased notably. At this stage SL increased markedly and MCL even more so, while BL remained relatively constant. After re-watering, the recovery of AN, gs and gm was rapid, although remaining below the levels of the control plants, while Vcmax fully regained control levels after three days of re-watering. MCL remained the main limitation factor irrespective of the degree of photosynthetic recovery. In conclusion, in our experiment MCL was the main photosynthetic limitation factor of R. delavayi under water stress and during the recovery phase, with the regulation of gm probably being the result of interactions between the environment and leaf anatomical features.

  16. Changes in photosynthesis, mesophyll conductance to CO2, and isoprenoid emissions in Populus nigra plants exposed to excess nickel

    International Nuclear Information System (INIS)

    Velikova, Violeta; Tsonev, Tsonko; Loreto, Francesco; Centritto, Mauro

    2011-01-01

    Poplar (Populus nigra) plants were grown hydroponically with 30 and 200 μM Ni (Ni 30 and Ni 200 ). Photosynthesis limitations and isoprenoid emissions were investigated in two leaf types (mature and developing). Ni stress significantly decreased photosynthesis, and this effect depended on the leaf Ni content, which was lower in mature than in developing leaves. The main limitations to photosynthesis were attributed to mesophyll conductance and metabolism impairment. In Ni-stressed developing leaves, isoprene emission was significantly stimulated. We attribute such stimulation to the lower chloroplastic [CO 2 ] than in control leaves. However chloroplastic [CO 2 ] did not control isoprene emission in mature leaves. Ni stress induced the emission of cis-β-ocimene in mature leaves, and of linalool in both leaf types. Induced biosynthesis and emission of isoprenoids reveal the onset of antioxidant processes that may also contribute to reduce Ni stress, especially in mature poplar leaves. - Graphical abstract: Visible damage caused by Ni treatment. 1 - Ni 0 (control plants); 2 - Ni 200 ; M = mature and D = developing Populus nigra leaves. Display Omitted Highlights: → We study the effect of Ni pollution on photosynthesis and isoprenoid emissions. → Ni stress significantly decreases photosynthesis. The main limitations are attributed to mesophyll conductance and metabolism impairment. → Constitutive isoprene emission was significantly stimulated in Ni-stressed leaves. Exposure to enhanced Ni concentration induces cis-beta-ocimene and linalool emissions. - The study reveals consequences of Ni stress on plant physiology, namely increasing diffusional limitation to photosynthesis and isoprenoid emissions.

  17. Analysis of the paired TCR α- and β-chains of single human T cells.

    Directory of Open Access Journals (Sweden)

    Song-Min Kim

    Full Text Available Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV and multiple sclerosis (MS. In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+ T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+ T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.

  18. Single cell biochemistry to visualize antigen presentation and drug resistance

    NARCIS (Netherlands)

    Griekspoor, Alexander Christiaan

    2006-01-01

    Many cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using antibodies to detect changes in protein levels, post-translational modifications, pairing with partner

  19. Whole-organism clone tracing using single-cell sequencing

    NARCIS (Netherlands)

    Alemany, Anna; Florescu, Maria; Baron, Chloé S; Peterson-Maduro, Josi; van Oudenaarden, Alexander

    2018-01-01

    Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and

  20. Single-centre experience of allogeneic haemopoietic stem cell ...

    African Journals Online (AJOL)

    Allogeneic haemopoietic stem cell transplant (Allo-HSCT) is used to treat a broad but well-defined range of paediatric conditions, most frequently in paediatric oncology for treatment intensification or salvage therapy for acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL). Allo-HSCT is also indicated in.

  1. Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

    Science.gov (United States)

    Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

    2014-01-01

    The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

  2. Quantifying the Traction Force of a Single Cell by Aligned Silicon Nanowire Array

    KAUST Repository

    Li, Zhou

    2009-10-14

    The physical behaviors of stationary cells, such as the morphology, motility, adhesion, anchorage, invasion and metastasis, are likely to be important for governing their biological characteristics. A change in the physical properties of mammalian cells could be an indication of disease. In this paper, we present a silicon-nanowire-array based technique for quantifying the mechanical behavior of single cells representing three distinct groups: normal mammalian cells, benign cells (L929), and malignant cells (HeLa). By culturing the cells on top of NW arrays, the maximum traction forces of two different tumor cells (HeLa, L929) have been measured by quantitatively analyzing the bending of the nanowires. The cancer cell exhibits a larger traction force than the normal cell by ∼20% for a HeLa cell and ∼50% for a L929 cell. The traction forces have been measured for the L929 cells and mechanocytes as a function of culture time. The relationship between cells extending area and their traction force has been investigated. Our study is likely important for studying the mechanical properties of single cells and their migration characteristics, possibly providing a new cellular level diagnostic technique. © 2009 American Chemical Society.

  3. Application of laser tweezers Raman spectroscopy techniques to the monitoring of single cell response to stimuli

    Science.gov (United States)

    Chan, James W.; Liu, Rui; Matthews, Dennis L.

    2012-06-01

    Laser tweezers Raman spectroscopy (LTRS) combines optical trapping with micro-Raman spectroscopy to enable label-free biochemical analysis of individual cells and small biological particles in suspension. The integration of the two technologies greatly simplifies the sample preparation and handling of suspension cells for spectroscopic analysis in physiologically meaningful conditions. In our group, LTRS has been used to study the effects of external perturbations, both chemical and mechanical, on the biochemistry of the cell. Single cell dynamics can be studied by performing longitudinal studies to continuously monitor the response of the cell as it interacts with its environment. The ability to carry out these measurements in-vitro makes LTRS an attractive tool for many biomedical applications. Here, we discuss the use of LTRS to study the response of cancer cells to chemotherapeutics and bacteria cells to antibiotics and show that the life cycle and apoptosis of the cells can be detected. These results show the promise of LTRS for drug discovery/screening, antibiotic susceptibility testing, and chemotherapy response monitoring applications. In separate experiments, we study the response of red blood cells to the mechanical forces imposed on the cell by the optical tweezers. A laser power dependent deoxygenation of the red blood cell in the single beam trap is reported. Normal, sickle cell, and fetal red blood cells have a different behavior that enables the discrimination of the cell types based on this mechanochemical response. These results show the potential utility of LTRS for diagnosing and studying red blood cell diseases.

  4. Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data.

    Science.gov (United States)

    Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong; Li, Mingyao; Zhang, Nancy R

    2017-11-02

    Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs

    NARCIS (Netherlands)

    Sanders, Ashley D; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Lansdorp, Peter M.

    The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange

  6. Single Cell HLA Matching Feasibility by Whole Genomic Amplification and Nested PCR

    Institute of Scientific and Technical Information of China (English)

    Xiao-hong Li; Fang-yin Meng

    2004-01-01

    @@ PCR based single-cell DNA analysis has been widely used in forensic science, preimplantation genetic diagnosis and so on. However, the original sample cannot be efficiently retrieved following single cell PCR, consequently the amount of information gained is limited. HLA system is too sophisticated that it is very hard to complete HLA typing by single cell. A Taq polymerase-based method using random primers to amplify whole genome termed as whole genome amplification (WGA) has demonstrated to be a useful method in increasing the copies of minimum sample. We establish a technique in this study to amplify HLA-A and HLA-B loci at same time in a single cell using WGA.

  7. Essentials of single-cell analysis concepts, applications and future prospects

    CERN Document Server

    Santra, Tuhin

    2016-01-01

    This book provides an overview of single-cell isolation, separation, injection, lysis and dynamics analysis as well as a study of their heterogeneity using different miniaturized devices. As an important part of single-cell analysis, different techniques including electroporation, microinjection, optical trapping, optoporation, rapid electrokinetic patterning and optoelectronic tweezers are described in detail. It presents different fluidic systems (e.g. continuous micro/nano-fluidic devices, microfluidic cytometry) and their integration with sensor technology, optical and hydrodynamic stretchers etc., and demonstrates the applications of single-cell analysis in systems biology, proteomics, genomics, epigenomics, cancer transcriptomics, metabolomics, biomedicine and drug delivery systems. It also discusses the future challenges for single-cell analysis, including the advantages and limitations. This book is enjoyable reading material while at the same time providing essential information to scientists in acad...

  8. Single cell proteomics in biomedicine: High-dimensional data acquisition, visualization, and analysis.

    Science.gov (United States)

    Su, Yapeng; Shi, Qihui; Wei, Wei

    2017-02-01

    New insights on cellular heterogeneity in the last decade provoke the development of a variety of single cell omics tools at a lightning pace. The resultant high-dimensional single cell data generated by these tools require new theoretical approaches and analytical algorithms for effective visualization and interpretation. In this review, we briefly survey the state-of-the-art single cell proteomic tools with a particular focus on data acquisition and quantification, followed by an elaboration of a number of statistical and computational approaches developed to date for dissecting the high-dimensional single cell data. The underlying assumptions, unique features, and limitations of the analytical methods with the designated biological questions they seek to answer will be discussed. Particular attention will be given to those information theoretical approaches that are anchored in a set of first principles of physics and can yield detailed (and often surprising) predictions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Hybrid confocal Raman fluorescence microscopy on single cells using semiconductor quantum dots

    NARCIS (Netherlands)

    van Manen, H.J.; Otto, Cornelis

    2007-01-01

    We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We

  10. A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells

    DEFF Research Database (Denmark)

    Yang, Tie; Bragheri, Francesca; Nava, Giovanni

    2016-01-01

    We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental ap...

  11. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

    2016-10-06

    Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Proximity-based differential single cell analysis of the niche to identify stem/progenitor cell regulators

    Science.gov (United States)

    Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Celso, Cristina Lo; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-fu; Scadden, David T

    2016-01-01

    SUMMARY Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on differential single-cell gene expression analysis of mesenchymal osteolineage cells close to and further removed from hematopoietic stem/progenitor cells to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. Amongst the genes which were preferentially expressed in proximal cells, we functionally examined three secreted or cell surface molecules not previously connected to HSPC biology: the secreted RNase Angiogenin, the cytokine IL18 and the adhesion molecule Embigin and discovered that all of these factors are HSPC quiescence regulators. Our proximity-based differential single cell approach therefore reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance understanding of microenvironmental regulation of stem cell function. PMID:27524439

  13. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction

    NARCIS (Netherlands)

    Mooijman, Dylan; Dey, Siddharth S; Boisset, Jean-Charles; Crosetto, Nicola; van Oudenaarden, Alexander

    2016-01-01

    The epigenetic DNA modification 5-hydroxymethylcytosine (5hmC) has crucial roles in development and gene regulation. Quantifying the abundance of this epigenetic mark at the single-cell level could enable us to understand its roles. We present a single-cell, genome-wide and strand-specific 5hmC

  14. RT-qPCR work-flow for single-cell data analysis

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Rusňáková, Vendula; Forootan, A.; Anděrová, Miroslava; Kubista, Mikael

    2013-01-01

    Roč. 59, č. 1 (2013), s. 80-88 ISSN 1046-2023 R&D Projects: GA MŠk(CZ) ME10052; GA ČR GAP303/10/1338 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378041 Keywords : RT-qPCR * Single-cell biology * Single-cell data analysis Subject RIV: EB - Genetics ; Molecular Biology; FH - Neurology (UEM-P) Impact factor: 3.221, year: 2013

  15. Rapid Evaluation of Power Degradation in Series Connection of Single Feeding Microsized Microbial Fuel Cells

    KAUST Repository

    Rojas, Jhonathan Prieto; Alqarni, Wejdan Mohammed Mofleh; Hussain, Muhammad Mustafa

    2014-01-01

    We have developed a sustainable, single feeding, microsized, air-cathode and membrane-free microbial fuel cells with a volume of 40 mu L each, which we have used for rapid evaluation of power generation and viability of a series array of three cells seeking higher voltage levels. Contrary to expectations, the achieved power density was modest (45 mWm(-3)), limited due to non-uniformities in assembly and the single-channel feeding system.

  16. Realignment process of actin stress fibers in single living cells studied by focused femtosecond laser irradiation

    OpenAIRE

    Yasukuni, Ryohei; Spitz, Jean-Alexis; Meallet-Renault, Rachel; Negishi, Takayuki; Tada, Takuji; Hosokawa, Yoichiroh; Asahi, Tsuyoshi; Shukunami, Chisa; Hiraki, Yuji; Masuhara, Hiroshi

    2007-01-01

    Three-dimensional dissection of a single actin stress fiber in a living cell was performed based on multi-photon absorption of a focused femtosecond laser pulse. The realignment process of an actin stress fiber was investigated after its direct cutting by a single-shot femtosecond laser pulse irradiation by high-speed transmission and fluorescence imaging methods. It was confirmed that mechanical force led by the femtosecond laser cutting propagates to entire cell through the cytockelton in a...

  17. Rapid Evaluation of Power Degradation in Series Connection of Single Feeding Microsized Microbial Fuel Cells

    KAUST Repository

    Rojas, Jhonathan Prieto

    2014-07-08

    We have developed a sustainable, single feeding, microsized, air-cathode and membrane-free microbial fuel cells with a volume of 40 mu L each, which we have used for rapid evaluation of power generation and viability of a series array of three cells seeking higher voltage levels. Contrary to expectations, the achieved power density was modest (45 mWm(-3)), limited due to non-uniformities in assembly and the single-channel feeding system.

  18. An Immunofluorescence-assisted Microfluidic Single Cell Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Tumour Cells Separated from Blood

    Directory of Open Access Journals (Sweden)

    Kazunori Hoshino

    2015-11-01

    matched the results from a few thousand cells. Some markers (e.g., ER, HER2 that are commonly used for cancer identification showed relatively large deviations in expres‐ sion levels. However, others (e.g., GRB7 showed devia‐ tions that are small enough to supplement single cell disease profiling.

  19. Tracing exogenous Gd and its effects in single Chang cells

    International Nuclear Information System (INIS)

    Altissimo, Matteo; Pascolo, Lorella; Delfino, Riccarda; Salome, Murielle; Lorusso, Vito

    2010-01-01

    X-ray fluorescence (XRF) is rapidly becoming one of the tools of choice in tracing the presence of both endogenous and exogenous chemical elements in biological samples. The sub-micron spatial resolution routinely obtainable at multi-keV energies at third generation light sources, combined with the high brilliance of the photon beam, allows mapping the presence of biologically relevant elements at sub-cell resolution by means of their fluorescent signature. The fluorescent signal also lends itself for a semi-quantitative analysis of the elements composing the specimen. In this work we employed synchrotron-based XRF to analyze two lines of cultured Chang cells. One of them was treated with a Gd-containing magnetic resonance imaging (MRI) contrast agents (CA), and the other was left untreated for control purposes. The experiments highlighted a peri-nuclear distribution of Gd inside the cells, as well as a distinct variation in the distribution and concentration of several elements (P, S, Cl, K, Ca and Fe), with respect to the control line.

  20. Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.

    Science.gov (United States)

    Liu, Mingshan; Liu, Yang; Di, Jiabo; Su, Zhe; Yang, Hong; Jiang, Beihai; Wang, Zaozao; Zhuang, Meng; Bai, Fan; Su, Xiangqian

    2017-11-23

    Colorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors. We sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels. A variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different. Our results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

  1. Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in C4-related metabolite fluxes and development.

    Directory of Open Access Journals (Sweden)

    Kalpana eManandhar-Shrestha

    2013-03-01

    Full Text Available As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second green revolution will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M and bundle sheath (BS chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts (cp. 74% have a known or predicted membrane association. 21 membrane proteins were 2-15 times more abundant in BS cells, while 36 proteins were more abundant in M cp envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of thirteen candidate genes. Cp association was confirmed using GFP labeling. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast

  2. Precise mass determination of single cell with cantilever-based microbiosensor system.

    Directory of Open Access Journals (Sweden)

    Bogdan Łabędź

    Full Text Available Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland, it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

  3. Precise mass determination of single cell with cantilever-based microbiosensor system.

    Science.gov (United States)

    Łabędź, Bogdan; Wańczyk, Aleksandra; Rajfur, Zenon

    2017-01-01

    Having determined the mass of a single cell of brewer yeast Saccharomyces cerevisiae by means of a microcantilever-based biosensor Cantisens CSR-801 (Concentris, Basel, Switzerland), it was found that its dry mass is 47,65 ± 1,05 pg. Found to be crucial in this mass determination was the cell position along the length of the cantilever. Moreover, calculations including cells positions on the cantilever provide a threefold better degree of accuracy than those which assume uniform mass distribution. We have also examined the influence of storage time on the single cell mass. Our results show that after 6 months there is an increase in the average mass of a single yeast cell.

  4. Effect of single-dose radiation on cell survival and growth hormone secretion by rat anterior pituitary cells

    International Nuclear Information System (INIS)

    Hochberg, Z.; Kuten, A.; Hertz, P.; Tatcher, M.; Kedar, A.; Benderly, A.

    1983-01-01

    Cranial irradiation has been shown to impair growth hormone secretion in children. In this study a cell culture of dispersed rat anterior pituitary cells was exposed to single doses of radiation in the range of 100 to 1500 rad. Survival curves were obtained for the different anterior pituitary cell lines, and growth hormone secretion was measured in the tissue culture medium. Both survival and growth hormone secretion curves showed an initial shoulder in the range of 0 to 300 rad, followed by a decline between 300 to 750 rad. It is concluded that growth hormone secreting acidophilic pituicytes are sensitive to radiation at single doses greater than 300 rad

  5. Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer

    DEFF Research Database (Denmark)

    Li, Yingrui; Xu, Xun; Song, Luting

    2012-01-01

    sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading...... to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively...

  6. Measurement of diffuse and specular reflections through single cell layers

    CSIR Research Space (South Africa)

    Karsten, AE

    2006-07-01

    Full Text Available ) • On average 50 000 new cases/year • LR at least • Male: 1 in 6 • Female: 1 in 7 Slide 4 © CSIR 2006 www.csir.co.za Background • Diabetes • High prevalence in SA • Not a notifiable disease • Indian: Av. 17% (11% - 30%) • Black...: 8% • White: 6% • Type II diabetes on the increase • Limp amputation • Research aimed at PDT and accelerated wound healing Slide 5 © CSIR 2006 www.csir.co.za Experimental work at UJ WS1Cell line Induce wound: sterile...

  7. Transverse mechanical properties of cell walls of single living plant cells probed by laser-generated acoustic waves.

    Science.gov (United States)

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

    Probing the mechanical properties of plant cell wall is crucial to understand tissue dynamics. However, the exact symmetry of the mechanical properties of this anisotropic fiber-reinforced composite remains uncertain. For this reason, biologically relevant measurements of the stiffness coefficients on individual living cells are a challenge. For this purpose, we have developed the single-cell optoacoustic nanoprobe (SCOPE) technique, which uses laser-generated acoustic waves to probe the stiffness, thickness and viscosity of live single-cell subcompartments. This all-optical technique offers a sub-micrometer lateral resolution, nanometer in-depth resolution, and allows the non-contact measurement of the mechanical properties of live turgid tissues without any assumption of mechanical symmetry. SCOPE experiments reveal that single-cell wall transverse stiffness in the direction perpendicular to the epidermis layer of onion cells is close to that of cellulose. This observation demonstrates that cellulose microfibrils are the main load-bearing structure in this direction, and suggests strong bonding of microfibrils by hemicelluloses. Altogether our measurement of the viscosity at high frequencies suggests that the rheology of the wall is dominated by glass-like dynamics. From a comparison with literature, we attribute this behavior to the influence of the pectin matrix. SCOPE's ability to unravel cell rheology and cell anisotropy defines a new class of experiments to enlighten cell nano-mechanics.

  8. Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

    Science.gov (United States)

    Andolfi, Laura; Bourkoula, Eugenia; Migliorini, Elisa; Palma, Anita; Pucer, Anja; Skrap, Miran; Scoles, Giacinto; Beltrami, Antonio Paolo; Cesselli, Daniela; Lazzarino, Marco

    2014-01-01

    Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

  9. The Effects of Nonuniform Illumination on the Electrical Performance of a Single Conventional Photovoltaic Cell

    Directory of Open Access Journals (Sweden)

    Damasen Ikwaba Paul

    2015-01-01

    Full Text Available Photovoltaic (PV concentrators are a promising approach for lowering PV electricity costs in the near future. However, most of the concentrators that are currently used for PV applications yield nonuniform flux profiles on the surface of a PV module which in turn reduces its electrical performance if the cells are serially connected. One way of overcoming this effect is the use of PV modules with isolated cells so that each cell generates current that is proportional to the energy flux absorbed. However, there are some cases where nonuniform illumination also exists in a single cell in an isolated cells PV module. This paper systematically studied the effect of nonuniform illumination on various cell performance parameters of a single monocrystalline standard PV cell at low and medium energy concentration ratios. Furthermore, the effect of orientation, size, and geometrical shapes of nonuniform illumination was also investigated. It was found that the effect of nonuniform illumination on various PV cell performance parameters of a single standard PV cell becomes noticeable at medium energy flux concentration whilst the location, size, and geometrical shape of nonuniform illumination have no effect on the performance parameters of the cell.

  10. Quantitative control of mitochondria transfer between live single cells using a microfluidic device

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

    Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

  11. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  12. Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

    Science.gov (United States)

    Breuer, Rebecca J; Bandyopadhyay, Arpan; O'Brien, Sofie A; Barnes, Aaron M T; Hunter, Ryan C; Hu, Wei-Shou; Dunny, Gary M

    2017-07-01

    In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.

  13. Sex chromosomes and germline transcriptomics explored by single-cell sequencing and RNA-tomography

    NARCIS (Netherlands)

    Vértesy, Ábel

    2018-01-01

    In our study of germ cell differentiation, we applied two recently developed technologies on the germline of various model organisms: single-cell mRNA sequencing and RNA-tomography. For the first time we could look at gene expression with such a high resolution, and this led us to discover the

  14. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software

    NARCIS (Netherlands)

    Kaiser, Matthias; Jug, Florian; Julou, Thomas; Deshpande, S.R.; Pfohl, Thomas; Silander, Olin K.; Myers, Gene; Van Nimwegen, Erik

    2018-01-01

    Much is still not understood about how gene regulatory interactions control cell fate decisions in single cells, in part due to the difficulty of directly observing gene regulatory processes in vivo. We introduce here a novel integrated setup consisting of a microfluidic chip and accompanying

  15. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

    NARCIS (Netherlands)

    van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

  16. Mechanics governs single-cell signaling and multi-cell robustness in biofilm infections

    Science.gov (United States)

    Gordon, Vernita

    In biofilms, bacteria and other microbes are embedded in extracellular polymers (EPS). Multiple types of EPS can be produced by a single bacterial strain - the reasons for this redundancy are not well-understood. Our work suggests that different polymers may confer distinct mechanical benefits. Our model organism is Pseudomonas aeruginosa, an opportunistic human pathogen that forms chronic biofilm infections associated with increased antibiotic resistance and evasion of the immune defense. Biofilms initiate when bacteria attach to a surface, sense the surface, and change their gene expression. Changes in gene expression are regulated by a chemical signal, cyclic-di-GMP. We find that one EPS material, called ``PEL,'' enhances surface sensing by increasing mechanical coupling of single bacteria to the surface. Measurements of bacterial motility suggest that PEL may increase frictional interactions between the surface and the bacteria. Consistent with this, we show that bacteria increase cyclic-di-GMP signaling in response to mechanical shear stress. Mechanosensing has long been known to be important to the function of cells in higher eukaryotes, but this is one of only a handful of studies showing that bacteria can sense and respond to mechanical forces. For the mature biofilm, the embedding polymer matrix can protect bacteria both chemically and mechanically. P. aeruginosa infections in the cystic fibrosis (CF) lung often last for decades, ample time for the infecting strain(s) to evolve. Production of another EPS material, alginate, is well-known to tend to increase over time in CF infections. Alginate chemically protects biofilms, but also makes them softer and weaker. Recently, it is being increasingly recognized that bacteria in chronic CF infections also evolve to increase PSL production. We use oscillatory bulk rheology to determine the unique contributions of EPS materials to biofilm mechanics. Unlike alginate, increased PSL stiffens biofilms. Increasing both

  17. Beta-Poisson model for single-cell RNA-seq data analyses.

    Science.gov (United States)

    Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Rantalainen, Mattias; Pawitan, Yudi

    2016-07-15

    Single-cell RNA-sequencing technology allows detection of gene expression at the single-cell level. One typical feature of the data is a bimodality in the cellular distribution even for highly expressed genes, primarily caused by a proportion of non-expressing cells. The standard and the over-dispersed gamma-Poisson models that are commonly used in bulk-cell RNA-sequencing are not able to capture this property. We introduce a beta-Poisson mixture model that can capture the bimodality of the single-cell gene expression distribution. We further integrate the model into the generalized linear model framework in order to perform differential expression analyses. The whole analytical procedure is called BPSC. The results from several real single-cell RNA-seq datasets indicate that ∼90% of the transcripts are well characterized by the beta-Poisson model; the model-fit from BPSC is better than the fit of the standard gamma-Poisson model in > 80% of the transcripts. Moreover, in differential expression analyses of simulated and real datasets, BPSC performs well against edgeR, a conventional method widely used in bulk-cell RNA-sequencing data, and against scde and MAST, two recent methods specifically designed for single-cell RNA-seq data. An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq data is available under GPL-3 license at https://github.com/nghiavtr/BPSC CONTACT: yudi.pawitan@ki.se or mattias.rantalainen@ki.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

    Science.gov (United States)

    Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

    2011-10-01

    A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

  19. The logic of single-cell projections from visual cortex.

    Science.gov (United States)

    Han, Yunyun; Kebschull, Justus M; Campbell, Robert A A; Cowan, Devon; Imhof, Fabia; Zador, Anthony M; Mrsic-Flogel, Thomas D

    2018-04-05

    Neocortical areas communicate through extensive axonal projections, but the logic of information transfer remains poorly understood, because the projections of individual neurons have not been systematically characterized. It is not known whether individual neurons send projections only to single cortical areas or distribute signals across multiple targets. Here we determine the projection patterns of 591 individual neurons in the mouse primary visual cortex using whole-brain fluorescence-based axonal tracing and high-throughput DNA sequencing of genetically barcoded neurons (MAPseq). Projections were highly diverse and divergent, collectively targeting at least 18 cortical and subcortical areas. Most neurons targeted multiple cortical areas, often in non-random combinations, suggesting that sub-classes of intracortical projection neurons exist. Our results indicate that the dominant mode of intracortical information transfer is not based on 'one neuron-one target area' mapping. Instead, signals carried by individual cortical neurons are shared across subsets of target areas, and thus concurrently contribute to multiple functional pathways.

  20. Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.

    Science.gov (United States)

    Ren, Wenwen; Lewandowski, Brian C; Watson, Jaime; Aihara, Eitaro; Iwatsuki, Ken; Bachmanov, Alexander A; Margolskee, Robert F; Jiang, Peihua

    2014-11-18

    Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

  1. Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm

    Directory of Open Access Journals (Sweden)

    Junjie Lu

    2018-04-01

    Full Text Available Differentiation of human pluripotent stem cells towards definitive endoderm (DE is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Keywords: hPSC, Differentiation, Definitive endoderm, Heterogeneity, Single cell, RNA sequencing

  2. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

    KAUST Repository

    Perozziello, Gerardo

    2015-12-11

    In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562). © 2015 Optical Society of America.

  3. Single-dose-response curves of murine gastrointestinal crypt stem cells

    International Nuclear Information System (INIS)

    Masuda, K.; Withers, H.R.; Mason, K.A.; Chen, K.Y.

    1977-01-01

    Dose-response curves for the reproductive capacity of crypt stem cells of murine colonic, jejunal, and gastric mucosae exposed in situ to multifractionated gamma ray exposures were analyzed and single-dose-survival curves of these cells were constructed. The following conclusions were drawn: (1) The single-dose-response curves bend downward over a dose range of approximately 200 to 1500 rad; (2) cell death seems to be due to nonrepairable damage at doses less than 250 rad for colon, and 220 rad for jejunum; (3) there are 21, 110, and 140 stem cells per crypt of gastric, colonic, and jejunal mucosa, respectively; and (4) jejunal stem cells are the most radiosensitive and gastric mucosal stem cells are the most resistant

  4. Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

    Directory of Open Access Journals (Sweden)

    Bragheri Francesca

    2017-08-01

    Full Text Available Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

  5. Impact of NBTI Aging on the Single-Event Upset of SRAM Cells

    CERN Document Server

    Bagatin, M; Gerardin, Simone; Paccagnella, Alessandro; Bagatin, Marta

    2010-01-01

    We analyzed the impact of negative bias temperature instability (NBTI) on the single-event upset rate of SRAM cells through experiments and SPICE simulations. We performed critical charge simulations introducing different degradation patterns in the cells, in three technology nodes, from 180 to 90 nm. The simulations results were checked with alpha-particle and heavy-ion irradiations on a 130-nm technology. Both simulations and experimental results show that NBTI degradation does not significantly affect the single-event upset SRAM cell rate as long as the parametric drift induced by aging is within 10\\%.

  6. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

    DEFF Research Database (Denmark)

    Perozziello, Gerardo; Candeloro, Patrizio; De Grazia, Antonio

    2016-01-01

    In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels-where the cells can flow one-by-one -, allowing single...... cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm...

  7. Quantifying cellular mechanics and adhesion in renal tubular injury using single cell force spectroscopy.

    Science.gov (United States)

    Siamantouras, Eleftherios; Hills, Claire E; Squires, Paul E; Liu, Kuo-Kang

    2016-05-01

    Tubulointerstitial fibrosis represents the major underlying pathology of diabetic nephropathy where loss of cell-to-cell adhesion is a critical step. To date, research has predominantly focussed on the loss of cell surface molecular binding events that include altered protein ligation. In the current study, atomic force microscopy single cell force spectroscopy (AFM-SCFS) was used to quantify changes in cellular stiffness and cell adhesion in TGF-β1 treated kidney cells of the human proximal tubule (HK2). AFM indentation of TGF-β1 treated HK2 cells showed a significant increase (42%) in the elastic modulus (stiffness) compared to control. Fluorescence microscopy confirmed that increased cell stiffness is accompanied by reorganization of the cytoskeleton. The corresponding changes in stiffness, due to F-actin rearrangement, affected the work of detachment by changing the separation distance between two adherent cells. Overall, our novel data quantitatively demonstrate a correlation between cellular elasticity, adhesion and early morphologic/phenotypic changes associated with tubular injury. Diabetes affects many patients worldwide. One of the long term problems is diabetic nephropathy. Here, the authors utilized atomic force microscopy single cell force spectroscopy (AFM- SCFS) to study cellular stiffness and cell adhesion after TGF1 treatment in human proximal tubule kidney cells. The findings would help further understand the overall disease mechanism in diabetic patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Opto-injection into single living cells by femtosecond near-infrared laser

    Science.gov (United States)

    Peng, Cheng

    This dissertation presents a novel technique to deliver membrane impermeable molecules into single living cells with the assistance of femtosecond (fs) near-infrared (NIR) laser pulses. This approach merges ultrafast laser technology with key biological, biomedical, and medical applications, such as gene transfection, gene therapy and drug delivery. This technique promises several major advantages, namely, very high transfection efficiency, high cell survival rate (≈100%) and fully preserved cell viabilities. It is also a promising method to deliver molecules into cells that are difficult or even completely resistant to established physical methods, such as microinjection by glass pipettes, electroporation, and biolistics. In this work, the system for fs NIR opto-injection was designed and built. Successful fs NIR opto-injection has been performed on several cell systems including single mammalian cells (bovine aortic endothelial cells), marine animal eggs (Spisula solidissima oocytes), and human cancer cells (fibrosarcoma HT1080) cultured in a tissue-like environment. The connections between laser parameters and cell responses were explored through further experiments and in-depth analyses, especially the relationship between dye uptake rate and incident laser intensity, and the relationship between pore size created on cell membranes and incident laser intensity. Dye uptake rate of the target cells was observed to depend on incident laser intensity. Pore size was found dependent on incident laser intensity. The conclusion was made that laser-induced breakdown and plasma-induced ablation in cell membrane are the physical principles that govern the process of fs NIR opto-injection.

  9. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  10. Single-Cell Transcriptomic Analysis Defines Heterogeneity and Transcriptional Dynamics in the Adult Neural Stem Cell Lineage

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    Ben W. Dulken

    2017-01-01

    Full Text Available Neural stem cells (NSCs in the adult mammalian brain serve as a reservoir for the generation of new neurons, oligodendrocytes, and astrocytes. Here, we use single-cell RNA sequencing to characterize adult NSC populations and examine the molecular identities and heterogeneity of in vivo NSC populations. We find that cells in the NSC lineage exist on a continuum through the processes of activation and differentiation. Interestingly, rare intermediate states with distinct molecular profiles can be identified and experimentally validated, and our analysis identifies putative surface markers and key intracellular regulators for these subpopulations of NSCs. Finally, using the power of single-cell profiling, we conduct a meta-analysis to compare in vivo NSCs and in vitro cultures, distinct fluorescence-activated cell sorting strategies, and different neurogenic niches. These data provide a resource for the field and contribute to an integrative understanding of the adult NSC lineage.

  11. A simple optical fiber device for quantitative fluorescence microscopy of single living cells

    OpenAIRE

    van Graft, M.; van Graft, Marja; Oosterhuis, B.; Oosterhuis, Bernard; van der Werf, Kees; de Grooth, B.G.; Greve, Jan

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultipl...

  12. Single Cell Oxygen Mapping (SCOM) by Scanning Electrochemical Microscopy Uncovers Heterogeneous Intracellular Oxygen Consumption

    OpenAIRE

    Santos, Carla Santana; Kowaltowski, Alicia J.; Bertotti, Mauro

    2017-01-01

    We developed a highly sensitive oxygen consumption scanning microscopy system using platinized platinum disc microelectrodes. The system is capable of reliably detecting single-cell respiration, responding to classical regulators of mitochondrial oxygen consumption activity as expected. Comparisons with commercial multi-cell oxygen detection systems show that the system has comparable errors (if not smaller), with the advantage of being able to monitor inter and intra-cell heterogeneity in ox...

  13. Raman tweezers spectroscopy of live, single red and white blood cells.

    Directory of Open Access Journals (Sweden)

    Aseefhali Bankapur

    Full Text Available An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC and white blood cells (WBC under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW. Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

  14. Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

    Science.gov (United States)

    Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

    2014-12-18

    Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

    Science.gov (United States)

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

    2015-03-30

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

  16. Single cell targeting using plasmon resonant gold-coated liposomes

    Science.gov (United States)

    Leung, Sarah J.; Romanowski, Marek

    2012-03-01

    We have developed an experimental system with the potential for the delivery and localized release of an encapsulated agent with high spatial and temporal resolution. We previously introduced liposome-supported plasmon resonant gold nanoshells; in this composite structure, the liposome allows for the encapsulation of substances, such as therapeutic agents, neurotransmitters, or growth factors, and the plasmon resonant structure facilitates the rapid release of encapsulated contents upon laser light illumination. More recently, we demonstrated that these gold-coated liposomes are capable of releasing their contents in a spectrally-controlled manner, where plasmon resonant nanoparticles only release content upon illumination with a wavelength of light matching their plasmon resonance band. We now show that this release mechanism can be used in a biological setting to deliver a peptide derivative of cholecystokinin to HEK293 cells overexpressing the CCK2 receptor. Using directed laser light, we may enable localized release from gold-coated liposomes to enable accurate perturbation of cellular functions in response to released compounds; this system may have possible applications in signaling pathways and drug discovery.

  17. Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

    Science.gov (United States)

    González-García, Mary-Paz; Pavelescu, Irina; Canela, Andrés; Sevillano, Xavier; Leehy, Katherine A; Nelson, Andrew D L; Ibañes, Marta; Shippen, Dorothy E; Blasco, Maria A; Caño-Delgado, Ana I

    2015-05-12

    Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Temporal dynamics and transcriptional control using single-cell gene expression analysis.

    Science.gov (United States)

    Kouno, Tsukasa; de Hoon, Michiel; Mar, Jessica C; Tomaru, Yasuhiro; Kawano, Mitsuoki; Carninci, Piero; Suzuki, Harukazu; Hayashizaki, Yoshihide; Shin, Jay W

    2013-01-01

    Changes in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event. Here we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages. We systematically measure temporal regulation of expression and variation by profiling 120 single cells at eight distinct time points, and infer highly controlled regulatory modules through which signaling operates with stochastic effects. This reveals dynamic and specific rewiring as a cellular strategy for differentiation. The integration of both positive and negative co-expression networks further identifies the proto-oncogene MYB as a network hinge to modulate both the pro- and anti-differentiation pathways. Compared to averaged cell populations, temporal single-cell expression profiling provides a much more powerful technique to probe for mechanistic insights underlying cellular differentiation. We believe that our approach will form the basis of novel strategies to study the regulation of transcription at a single-cell level.

  19. Single cells for forensic DNA analysis--from evidence material to test tube.

    Science.gov (United States)

    Brück, Simon; Evers, Heidrun; Heidorn, Frank; Müller, Ute; Kilper, Roland; Verhoff, Marcel A

    2011-01-01

    The purpose of this project was to develop a method that, while providing morphological quality control, allows single cells to be obtained from the surfaces of various evidence materials and be made available for DNA analysis in cases where only small amounts of cell material are present or where only mixed traces are found. With the SteREO Lumar.V12 stereomicroscope and UV unit from Zeiss, it was possible to detect and assess single epithelial cells on the surfaces of various objects (e.g., glass, plastic, metal). A digitally operated micromanipulator developed by aura optik was used to lift a single cell from the surface of evidence material and to transfer it to a conventional PCR tube or to an AmpliGrid(®) from Advalytix. The actual lifting of the cells was performed with microglobes that acted as carriers. The microglobes were held with microtweezers and were transferred to the DNA analysis receptacles along with the adhering cells. In a next step, the PCR can be carried out in this receptacle without removing the microglobe. Our method allows a single cell to be isolated directly from evidence material and be made available for forensic DNA analysis. © 2010 American Academy of Forensic Sciences.

  20. Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing.

    Science.gov (United States)

    Hochgerner, Hannah; Zeisel, Amit; Lönnerberg, Peter; Linnarsson, Sten

    2018-02-01

    The dentate gyrus of the hippocampus is a brain region in which neurogenesis persists into adulthood; however, the relationship between developmental and adult dentate gyrus neurogenesis has not been examined in detail. Here we used single-cell RNA sequencing to reveal the molecular dynamics and diversity of dentate gyrus cell types in perinatal, juvenile, and adult mice. We found distinct quiescent and proliferating progenitor cell types, linked by transient intermediate states to neuroblast stages and fully mature granule cells. We observed shifts in the molecular identity of quiescent and proliferating radial glia and granule cells during the postnatal period that were then maintained through adult stages. In contrast, intermediate progenitor cells, neuroblasts, and immature granule cells were nearly indistinguishable at all ages. These findings demonstrate the fundamental similarity of postnatal and adult neurogenesis in the hippocampus and pinpoint the early postnatal transformation of radial glia from embryonic progenitors to adult quiescent stem cells.

  1. POOR HEMOPOIETIC STEM CELL MOBILIZERS IN MULTIPLE MYELOMA : A SINGLE INSTITUTION EXPERIENCE

    Directory of Open Access Journals (Sweden)

    Guillermo Jose Ruiz-Delgado

    2010-06-01

    Full Text Available In a single institution, in a group of 28 myeloma patients deemed eligible for autologous transplant, stem cell mobilization was attempted using filgrastim: 26 individuals were given 31 autografts employing 1-4 (median three apheresis sessions, to obtain a target stem cell dose of 1 x 106 CD34 viable cells / Kg of the recipient. The median number of grafted CD34 cells was 7.56 x 106  / Kg of the recipient; the range being 0.92 to 14.8.  By defining as poor mobilizers individuals in which a cell collection of 1 x 106 CD34 viable cells / Kg was better (80% at 80 months than those grafted with < 1 x 106 CD34 viable cells / Kg (67% at 76 months. Methods to improve stem cell mobilization are needed and may result in obtaining better results when autografting multiple myeloma patients.

  2. The Caenorhabditis elegans Q neuroblasts: A powerful system to study cell migration at single-cell resolution in vivo.

    Science.gov (United States)

    Rella, Lorenzo; Fernandes Póvoa, Euclides E; Korswagen, Hendrik C

    2016-04-01

    During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this article, we discuss the Caenorhabditis elegans Q neuroblasts and their descendants as a tool to study cell migration at single-cell resolution in vivo. The highly stereotypical migration of these cells provides a powerful system to study the dynamic cytoskeletal processes that drive migration as well as the evolutionarily conserved signaling pathways (including different Wnt signaling cascades) that guide the cells along their specific trajectories. Here, we provide an overview of what is currently known about Q neuroblast migration and highlight the live-cell imaging, genome editing, and quantitative gene expression techniques that have been developed to study this process. © 2016 Wiley Periodicals, Inc.

  3. Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle

    Directory of Open Access Journals (Sweden)

    Soloviev Mikhail

    2010-09-01

    Full Text Available Abstract Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.

  4. The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

    Science.gov (United States)

    Fujiwara, Makoto T; Yasuzawa, Mana; Kojo, Kei H; Niwa, Yasuo; Abe, Tomoko; Yoshida, Shigeo; Nakano, Takeshi; Itoh, Ryuuichi D

    2018-01-01

    of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.

  5. A portable system powered with hydrogen and one single air-breathing PEM fuel cell

    International Nuclear Information System (INIS)

    Fernández-Moreno, J.; Guelbenzu, G.; Martín, A.J.; Folgado, M.A.; Ferreira-Aparicio, P.; Chaparro, A.M.

    2013-01-01

    Highlights: • A portable system based on hydrogen and single air breathing PEM fuel cell. • Control electronics designed for low single cell voltage (0.5–0.8 V). • Forced air convection and anode purging required to help water management. • Application consisting of a propeller able to display a luminous message. • Up to 20 h autonomy with continuous 1.1 W consumption, using 1 g H 2 . - Abstract: A portable system for power generation based on hydrogen and a single proton exchange membrane fuel cell (PEMFC) has been built and operated. The fuel cell is fed in the anode with hydrogen stored in a metal hydrides cartridge, and in the cathode with oxygen from quiescent ambient air (‘air breathing’). The control electronics of the system performs DC–DC conversion from the low voltage (0.5–0.8 V) and high current output (200–300 mA cm −2 ) of the single fuel cell, up to 3.3 V to power an electronic application. System components assist fuel cell operation, including an electronic valve for anode purging, a fan in front of the open cathode, two supercapacitors for auxiliary power requirements, four LED lights, and a display screen. The influence of the system components on fuel cell behaviour is analyzed. The cathode fan and anodic purging help excess water removal from the electrodes leading to steadier cell response at the expense of extra power consumption. The power system is able to provide above 1 W DC electricity to an external application during 20 h using 1 g of H 2 . An application consisting of a propeller able to display a luminous message is chosen to test system. It is shown that one single air breathing PEM fuel cell powered with hydrogen may provide high energy density and autonomy for portable applications

  6. A quantitative comparison of single-cell whole genome amplification methods.

    Directory of Open Access Journals (Sweden)

    Charles F A de Bourcy

    Full Text Available Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA, Multiple Annealing and Looping Based Amplification Cycles (MALBAC, and the PicoPLEX single-cell WGA kit (NEB-WGA. We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

  7. visnormsc: A Graphical User Interface to Normalize Single-cell RNA Sequencing Data.

    Science.gov (United States)

    Tang, Lijun; Zhou, Nan

    2017-12-26

    Single-cell RNA sequencing (RNA-seq) allows the analysis of gene expression with high resolution. The intrinsic defects of this promising technology imports technical noise into the single-cell RNA-seq data, increasing the difficulty of accurate downstream inference. Normalization is a crucial step in single-cell RNA-seq data pre-processing. SCnorm is an accurate and efficient method that can be used for this purpose. An R implementation of this method is currently available. On one hand, the R package possesses many excellent features from R. On the other hand, R programming ability is required, which prevents the biologists who lack the skills from learning to use it quickly. To make this method more user-friendly, we developed a graphical user interface, visnormsc, for normalization of single-cell RNA-seq data. It is implemented in Python and is freely available at https://github.com/solo7773/visnormsc . Although visnormsc is based on the existing method, it contributes to this field by offering a user-friendly alternative. The out-of-the-box and cross-platform features make visnormsc easy to learn and to use. It is expected to serve biologists by simplifying single-cell RNA-seq normalization.

  8. Microelectromechanical System-Based Sensing Arrays for Comparative in Vitro Nanotoxicity Assessment at Single Cell and Small Cell-Population Using Electrochemical Impedance Spectroscopy.

    Science.gov (United States)

    Shah, Pratikkumar; Zhu, Xuena; Zhang, Xueji; He, Jin; Li, Chen-zhong

    2016-03-09

    The traditional in vitro nanotoxicity assessment approaches are conducted on a monolayer of cell culture. However, to study a cell response without interference from the neighbor cells, a single cell study is necessary; especially in cases of neuronal, cancerous, and stem cells, wherein an individual cell's fate is often not explained by the whole cell population. Nonetheless, a single cell does not mimic the actual in vivo environment and lacks important information regarding cell communication with its microenvironment. Both a single cell and a cell population provide important and complementary information about cells' behaviors. In this research, we explored nanotoxicity assessment on a single cell and a small cell population using electrochemical impedance spectroscopy and a microelectromechanical system (MEMS) device. We demonstrated a controlled capture of PC12 cells in different-sized microwells (to capture a different number of cells) using a combined method of surface functionalization and dielectrophoresis. The present approach provides a rapid nanotoxicity response as compared to other conventional approaches. This is the first study, to our knowledge, which demonstrates a comparative response of a single cell and small cell colonies on the same MEMS platform, when exposed to metaloxide nanoparticles. We demonstrated that the microenvironment of a cell is also accountable for cells' behaviors and their responses to nanomaterials. The results of this experimental study open up a new hypothesis to be tested for identifying the role of cell communication in spreading toxicity in a cell population.

  9. Microarray Expression Analyses of Arabidopsis Guard Cells and Isolation of a Recessive Abscisic Acid Hypersensitive Protein Phosphatase 2C MutantW⃞

    Science.gov (United States)

    Leonhardt, Nathalie; Kwak, June M.; Robert, Nadia; Waner, David; Leonhardt, Guillaume; Schroeder, Julian I.

    2004-01-01

    Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell–expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription–PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)–regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type–specific genomic scale analyses of gene function. PMID:14973164

  10. A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

    Directory of Open Access Journals (Sweden)

    Birte Möhlendick

    Full Text Available Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH of single cells. The protocol is based on an established adapter-linker PCR (WGAM and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost- effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

  11. Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level

    Science.gov (United States)

    Swiers, Gemma; Baumann, Claudia; O'Rourke, John; Giannoulatou, Eleni; Taylor, Stephen; Joshi, Anagha; Moignard, Victoria; Pina, Cristina; Bee, Thomas; Kokkaliaris, Konstantinos D.; Yoshimoto, Momoko; Yoder, Mervin C.; Frampton, Jon; Schroeder, Timm; Enver, Tariq; Göttgens, Berthold; de Bruijn, Marella F. T. R.

    2013-12-01

    Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP+ HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.

  12. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  13. Detecting infrared luminescence and non-chemical signaling of living cells: single cell mid-IR spectroscopy in cryogenic environments

    Science.gov (United States)

    Pereverzev, Sergey

    2017-02-01

    Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.

  14. Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

    Science.gov (United States)

    Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

    2017-12-21

    High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

  15. Mechanisms of gravitropism in single-celled systems

    Science.gov (United States)

    Greuel, Nicole; Braun, Markus; Hauslage, Jens; Wiemann, Katharina

    Physiological processes in plants are influenced by a variety of external stimuli. Gravity is the only constant factor that provides plants with reliable information for their orientation. Gravity-oriented growth responses, called gravitropism, enable plants to adapt to a diversity of habitats on Earth and to survive changing environmental conditions. For instance, the ability to respond gravitropically prevents crop, flattened by a windstorm, from decay. Even small deviations from the genetically programmed set-point angle of plant organs are recognized by specialized cells, the statocytes, in which dense particles, the statoliths, sediment in the direction of gravity and activate gravity sensors - membrane bound gravity-receptor proteins. Activation of receptor proteins creates a physiological signal that initiates a stimulus-specific signal transduction cascade causing the gravitropic response. To unravel the gravitropic signalling pathways in plant statocytes, our research focused on a unicellular model system, the rhizoid of the green alga Chara. Experiments under microgravity conditions during sounding-rocket and parabolic plane flights have shown that the actin cytoskeleton is a key element of the gravityinduced statolith-sedimentation process in characean rhizoids. Actomyosin, consisting of a dense meshwork of mainly axially oriented actin microfilaments and motor proteins (myosins), actively guides sedimenting statoliths to gravisensitive plasma membrane areas where gravireceptor molecules are exclusively located. TEXUS and MAXUS sounding rocket missions were performed to determine the threshold acceleration level (membrane-bound gravireceptor in characean rhizoids. The results contradict the classical model of a mechanoreceptor that is activated by the pressure exerted by sedimented statoliths. Instead, the experiments provide evidence that graviperception depends on direct interactions between statoliths and a yet unknown gravireceptor.Graviperception in

  16. Prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Che K

    2017-02-01

    Full Text Available Keying Che,1,* Yang Zhao,2,3,* Xiao Qu,1 Zhaofei Pang,1 Yang Ni,4 Tiehong Zhang,4 Jiajun Du,1,5 Hongchang Shen4 1Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 2Department of Breast Surgery, Key Laboratory of Breast Cancer in Shanghai, Collaborative Innovation Center of Cancer Medicine, Fudan University Shanghai Cancer Center, 3Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 4Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, 5Department of Thoracic Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, People’s Republic of China *These authors contributed equally to this work Purpose: Gastric carcinoma (GC is a highly aggressive cancer and one of the leading causes of cancer-related deaths worldwide. Histopathological evaluation pertaining to invasiveness is likely to provide additional information in relation to patient outcome. In this study, we aimed to evaluate the prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma.Materials and methods: Hematoxylin and eosin-stained slides generated from 296 gastric adenocarcinoma patients with full clinical and pathological and follow-up information were systematically reviewed. The patients were grouped on the basis of tumor budding, single cell invasion, large cell invasion, mitotic count, and fibrosis. The association between histopathological parameters, different classification systems, and overall survival (OS was statistically analyzed.Results: Among the 296 cases that were analyzed, high-grade tumor budding was observed in 49.0% (145 of them. Single cell invasion and large cell invasion were observed in 62.8% (186 and 16.9% (50 of the cases, respectively. Following univariate analysis, patients with high-grade tumor budding had shorter OS than those with low-grade tumor budding (hazard ratio [HR]: 2.260, P<0

  17. Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

    International Nuclear Information System (INIS)

    Lipp, A. M.

    2012-01-01

    Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

  18. An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

    Science.gov (United States)

    Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

    2012-12-01

    Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

  19. The Fate of a Normal Human Cell Traversed by a Single Charged Particle

    Science.gov (United States)

    Fournier, C.; Zahnreich, S.; Kraft, D.; Friedrich, T.; Voss, K.-O.; Durante, M.; Ritter, S.

    2012-01-01

    The long-term “fate” of normal human cells after single hits of charged particles is one of the oldest unsolved issues in radiation protection and cellular radiobiology. Using a high-precision heavy-ion microbeam we could target normal human fibroblasts with exactly one or five carbon ions and measured the early cytogenetic damage and the late behaviour using single-cell cloning. Around 70% of the first cycle cells presented visible aberrations in mFISH after a single ion traversal, and about 5% of the cells were still able to form colonies. In one third of selected high-proliferative colonies we observed clonal (radiation-induced) aberrations. Terminal differentiation and markers of senescence (PCNA, p16) in the descendants of cells traversed by one carbon ion occurred earlier than in controls, but no evidence of radiation-induced chromosomal instability was found. We conclude that cells surviving single-ion traversal, often carrying clonal chromosome aberrations, undergo accelerated senescence but maintain chromosomal stability. PMID:22966418

  20. Single-dose and fractionated irradiation of four human lung cancer cell lines in vitro

    International Nuclear Information System (INIS)

    Brodin, O.; Lennartsson, L.; Nilsson, S.

    1991-01-01

    Four established human lung cancer cell lines were exposed to single-dose irradiation. The survival curves of 2 small cell lung carcinomas (SCLC) were characterized by a limited capacity for repair with small and moderate shoulders with extrapolation numbers (n) of 1.05 and 1.60 respectively. Two non-small cell lung carcinoma (NSCLC) cell lines, one squamous cell (SQCLC) and one large cell (LCLC) had large shoulders with n-values of 73 and 15 respectively. The radiosensitivity when measured as D 0 did not, however, differ as much from cell line to cell line, with values from 1.22 to 1.65. The surviving fraction after 2 Gy (SF2) was 0.24 and 0.42 respectively in the SCLC cell lines and 0.90 and 0.88 respectively in the NSCLC cell lines. Fractionated irradiation delivered according to 3 different schedules was also investigated. All the schedules delivered a total dose of 10 Gy in 5 days and were applied in 1, 2 and 5 Gy dose fractions respectively. Survival followed the pattern found after single-dose irradiation; it was lowest in the SCLC cell line with the lowest SF and highest in the two NSCLC cell lines. In the SCLC cell lines all schedules were approximately equally efficient. In the LCLC and in the SQCLC cell lines, the 5 Gy schedule killed more cells than the 1 and 2 Gy schedules. The results indicate that the size of the shoulder of the survival curve is essential when choosing the most tumoricidal fractionation schedule. (orig.)

  1. Microchip screening platform for single cell assessment of NK cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Karolin eGuldevall

    2016-04-01

    Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  2. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    Science.gov (United States)

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  3. Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

    Directory of Open Access Journals (Sweden)

    Zhenzhen eQiao

    2013-11-01

    Full Text Available Plant root is an organ composed of multiple cell types with different functions. This multicellular complexity limits our understanding of root biology because –omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e. uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes, the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous treatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000 mg of plant cells from 60 root systems. Using soybean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soybean root hair specific between preparations of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach.

  4. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Adhesion and migration of CHO cells on micropatterned single layer graphene

    Science.gov (United States)

    Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

    2017-06-01

    Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

  6. Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

    Directory of Open Access Journals (Sweden)

    Yuting Guo

    2017-07-01

    Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

  7. Electrochemical Quantification of Extracellular Local H2O2 Kinetics Originating from Single Cells.

    Science.gov (United States)

    Bozem, Monika; Knapp, Phillip; Mirčeski, Valentin; Slowik, Ewa J; Bogeski, Ivan; Kappl, Reinhard; Heinemann, Christian; Hoth, Markus

    2017-05-15

    H 2 O 2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local H 2 O 2 concentrations ([H 2 O 2 ]) originating from single cells is required. Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H 2 O 2 ] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H 2 O 2 ] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H 2 O 2 ] 5-8 μm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H 2 O 2 to an unstimulated MC, the local [H 2 O 2 ] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H 2 O 2 is evenly distributed around the producing cell and can still be detected up to 30 μm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex ® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H 2 O 2 separately. Local extracellular [H 2 O 2 ] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 00, 000-000.

  8. Using measures of single-cell physiology and physiological state to understand organismic aging.

    Science.gov (United States)

    Mendenhall, Alexander; Driscoll, Monica; Brent, Roger

    2016-02-01

    Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  9. Guard cell zeaxanthin tracks photosynthetically active radiation and stomatal apertures in Vicia faba leaves

    International Nuclear Information System (INIS)

    Srivastava, A.; Zeiger, E.

    1995-01-01

    Zeaxanthin, antheraxanthin and violaxanthin concentrations in guard cells from sonicated abaxial epidermal peels of Vicia faba were measured from dawn to dusk, and compared with concentrations in mesophyll tissue of the same leaves. Measured changes in guard cell zeaxanthin and violaxanthin concentrations indicate that guard cells operate the xanthophyll cycle throughout the day. Mesophyll tissue had no detectable zeaxanthin at dawn, whereas guard cells had 30–50 mmol mol −1 chlorophyll a+b. On a chlorophyll basis, maximal zeaxanthin levels were 3–4 fold higher in guard cells than in mesophyll cells. Zeaxanthin concentrations tracked levels of photosynthetically active radiation (PAR) in both mesophyll and guard cells. In the mesophyll, most of the zeaxanthin changes occurred in mid-morning and mid-afternoon. In guard cells, zeaxanthin concentrations changed nearly linearly with PAR in the early morning and late afternoon, and closely tracked PAR levels throughout the day. Guard cell zeaxanthin concentrations were also closely correlated with stomatal apertures. The close relationship between zeaxanthin concentrations and PAR levels in guard cells indicates that zeaxanthin is well suited to function as a molecular photosensor in stomatal movements. (author)

  10. [Prediction of the molecular response to pertubations from single cell measurements].

    Science.gov (United States)

    Remacle, Françoise; Levine, Raphael D

    2014-12-01

    The response of protein signalization networks to perturbations is analysed from single cell measurements. This experimental approach allows characterizing the fluctuations in protein expression levels from cell to cell. The analysis is based on an information theoretic approach grounded in thermodynamics leading to a quantitative version of Le Chatelier principle which allows to predict the molecular response. Two systems are investigated: human macrophages subjected to lipopolysaccharide challenge, analogous to the immune response against Gram-negative bacteria and the response of the proteins involved in the mTOR signalizing network of GBM cancer cells to changes in partial oxygen pressure. © 2014 médecine/sciences – Inserm.

  11. Single cell adhesion strength assessed with variable-angle total internal reflection fluorescence microscopy

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    Marcelina Cardoso Dos Santos

    2017-06-01

    Full Text Available We propose a new strategy to evaluate adhesion strength at the single cell level. This approach involves variable-angle total internal reflection fluorescence microscopy to monitor in real time the topography of cell membranes, i.e. a map of the membrane/substrate separation distance. According to the Boltzmann distribution, both potential energy profile and dissociation energy related to the interactions between the cell membrane and the substrate were determined from the membrane topography. We have highlighted on glass substrates coated with poly-L-lysine and fibronectin, that the dissociation energy is a reliable parameter to quantify the adhesion strength of MDA-MB-231 motile cells.

  12. Single Nisoldipine-Sensitive Calcium Channels in Smooth Muscle Cells Isolated from Rabbit Mesenteric Artery

    Science.gov (United States)

    Worley, Jennings F.; Deitmer, Joachim W.; Nelson, Mark T.

    1986-08-01

    Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types of channels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.

  13. Single-bacterium nanomechanics in biomedicine: unravelling the dynamics of bacterial cells

    International Nuclear Information System (INIS)

    Aguayo, S; Bozec, L; Donos, N; Spratt, D

    2015-01-01

    The use of the atomic force microscope (AFM) in microbiology has progressed significantly throughout the years since its first application as a high-resolution imaging instrument. Modern AFM setups are capable of characterizing the nanomechanical behaviour of bacterial cells at both the cellular and molecular levels, where elastic properties and adhesion forces of single bacterium cells can be examined under different experimental conditions. Considering that bacterial and biofilm-mediated infections continue to challenge the biomedical field, it is important to understand the biophysical events leading towards bacterial adhesion and colonization on both biological and non-biological substrates. The purpose of this review is to present the latest findings concerning the field of single-bacterium nanomechanics, and discuss future trends and applications of nanoindentation and single-cell force spectroscopy techniques in biomedicine. (topical review)

  14. Comparison of whole genome amplification techniques for human single cell exome sequencing.

    Science.gov (United States)

    Borgström, Erik; Paterlini, Marta; Mold, Jeff E; Frisen, Jonas; Lundeberg, Joakim

    2017-01-01

    Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.

  15. Suppressive effects of primed eosinophils on single epicutaneous sensitization through regulation of dermal dendritic cells.

    Science.gov (United States)

    Lin, Jing-Yi; Ta, Yng-Cun; Liu, I-Lin; Chen, Hsi-Wen; Wang, Li-Fang

    2016-07-01

    Eosinophils are multifunctional innate immune cells involved in many aspects of innate and adaptive immunity. Epicutaneous sensitization with protein allergen is an important sensitization route for atopic dermatitis. In this study, using a murine single protein-patch model, we show that eosinophils of a primed status accumulate in draining lymph nodes following single epicutaneous sensitization. Further, depletion of eosinophils results in enhancement of the induced Th1/Th2 immune responses, whereas IL-5-induced hypereosinophilia suppresses these responses. Mechanistically, primed eosinophils cause a reduction in the numbers and activation status of dermal dendritic cells in draining lymph nodes. Collectively, these results demonstrate that primed eosinophils exert suppressive effects on single epicutaneous sensitization through regulation of dermal dendritic cells. Thus, these findings highlight the critical roles of eosinophils in the pathogenesis of atopic dermatitis with important clinical implications for the prevention of allergen sensitization. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

    Directory of Open Access Journals (Sweden)

    Anetzberger Claudia

    2012-09-01

    Full Text Available Abstract Background Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection and exoproteolytic activity (fluorescence of a promoter::gfp fusion, in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  17. Single cell biology beyond the era of antibodies: relevance, challenges, and promises in biomedical research.

    Science.gov (United States)

    Abraham, Parvin; Maliekal, Tessy Thomas

    2017-04-01

    Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.

  18. An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

    Science.gov (United States)

    Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

    2018-05-17

    Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals.

    Science.gov (United States)

    Anetzberger, Claudia; Schell, Ursula; Jung, Kirsten

    2012-09-18

    Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  20. [Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

    Science.gov (United States)

    An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

    2012-12-01

    We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

  1. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-01-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  2. Modifying infrared scattering effects of single yeast cells with plasmonic metal mesh

    Science.gov (United States)

    Malone, Marvin A.; Prakash, Suraj; Heer, Joseph M.; Corwin, Lloyd D.; Cilwa, Katherine E.; Coe, James V.

    2010-11-01

    The scattering effects in the infrared (IR) spectra of single, isolated bread yeast cells (Saccharomyces cerevisiae) on a ZnSe substrate and in metal microchannels have been probed by Fourier transform infrared imaging microspectroscopy. Absolute extinction [(3.4±0.6)×10-7 cm2 at 3178 cm-1], scattering, and absorption cross sections for a single yeast cell and a vibrational absorption spectrum have been determined by comparing it to the scattering properties of single, isolated, latex microspheres (polystyrene, 5.0 μm in diameter) on ZnSe, which are well modeled by the Mie scattering theory. Single yeast cells were then placed into the holes of the IR plasmonic mesh, i.e., metal films with arrays of subwavelength holes, yielding "scatter-free" IR absorption spectra, which have undistorted vibrational lineshapes and a rising generic IR absorption baseline. Absolute extinction, scattering, and absorption spectral profiles were determined for a single, ellipsoidal yeast cell to characterize the interplay of these effects.

  3. Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior.

    Science.gov (United States)

    Kobayashi, Kyogo; Nakano, Shunji; Amano, Mutsuki; Tsuboi, Daisuke; Nishioka, Tomoki; Ikeda, Shingo; Yokoyama, Genta; Kaibuchi, Kozo; Mori, Ikue

    2016-01-05

    Unveiling the molecular and cellular mechanisms underlying memory has been a challenge for the past few decades. Although synaptic plasticity is proven to be essential for memory formation, the significance of "single-cell memory" still remains elusive. Here, we exploited a primary culture system for the analysis of C. elegans neurons and show that a single thermosensory neuron has an ability to form, retain, and reset a temperature memory. Genetic and proteomic analyses found that the expression of the single-cell memory exhibits inter-individual variability, which is controlled by the evolutionarily conserved CaMKI/IV and Raf pathway. The variable responses of a sensory neuron influenced the neural activity of downstream interneurons, suggesting that modulation of the sensory neurons ultimately determines the behavioral output in C. elegans. Our results provide proof of single-cell memory and suggest that the individual differences in neural responses at the single-cell level can confer individuality. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

    Science.gov (United States)

    Maglica, Željka; Özdemir, Emre; McKinney, John D

    2015-02-17

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

  5. Performance features of 22-cell, 19Ah single pressure vessel nickel hydrogen battery

    Energy Technology Data Exchange (ETDEWEB)

    Rao, G.M.; Vaidyanathan, H.

    1996-02-01

    Two 22-cells 19Ah Nickel-Hydrogen (Ni-H2) Single Pressure Vessel (SPV) Qual batteries, one each from EPI/Joplin and EPI/Butler, were designed and procured. The two batteries differ in the cell encapsulation technology, stack preload, and activation procedure. Both the Butler and Joplin batteries met the specified requirements when subjected to qualification testing and completed 2100 and 1300 LEO cycles respectively, with nominal performance. This paper discusses advantages, design features, testing procedures, and results of the two single pressure vessel Ni-H2 batteries.

  6. Performance features of 22-cell, 19Ah single pressure vessel nickel hydrogen battery

    Science.gov (United States)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    1996-01-01

    Two 22-cells 19Ah Nickel-Hydrogen (Ni-H2) Single Pressure Vessel (SPV) Qual batteries, one each from EPI/Joplin and EPI/Butler, were designed and procured. The two batteries differ in the cell encapsulation technology, stack preload, and activation procedure. Both the Butler and Joplin batteries met the specified requirements when subjected to qualification testing and completed 2100 and 1300 LEO cycles respectively, with nominal performance. This paper discusses advantages, design features, testing procedures, and results of the two single pressure vessel Ni-H2 batteries.

  7. Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

    Science.gov (United States)

    Ullal, Adeeti V; Weissleder, Ralph

    2015-01-01

    We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

  8. Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

    Directory of Open Access Journals (Sweden)

    Yann Marcy

    2007-09-01

    Full Text Available Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

  9. Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

    Science.gov (United States)

    Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

    2014-05-01

    An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

  10. Development of Single Cell Raman Spectroscopy for Cancer Screening and Therapy Monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Chan, James W. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2009-02-24

    The overall goal of this project was to develop a new technology for cancer detection based on single cell laser tweezers Raman spectroscopy (LTRS). This method has the potential to improve the detection of cancer characteristics in single cells by acquiring cellular spectral markers that reflect the intrinsic biology of the cell. These spectral biomarkers are a new form of molecular signatures in the field of cancer research that may hold promise in accurately identifying and diagnosing cancer and measuring pati