Sample records for single-cell gel comet

  1. Viable human buccal mucosa cells do not yield typical nucleoids: impacts on the single-cell gel electrophoresis/Comet assay. (United States)

    Pinhal, Danillo; Gontijo, Alisson Marques de Miranda Cabral; Reyes, Victor Alexis Valenzuela; Salvadori, Daisy Maria Favero


    Buccal mucosa (BM) cells have been used in human biomonitoring studies for detecting DNA adducts and chromosomal damage in an epithelial cell population. In the present study, we have investigated if human BM cells are suitable for use in the single-cell gel electrophoresis (SCGE)/Comet assay as an approach for estimating the exposure of epithelial cells to DNA-damaging agents. Our results indicate that only a few cells from BM cell samples yield comets that can be analyzed by current methods, and that the yield of cells with comets is independent of the percentage of viable BM cells in the sample. Data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggest that most of the BM cells that do form comets are probably leukocytes. Moreover, by reevaluating specific cells after running the Comet assay, we found that viable epithelial BM cells give rise to atypical comets that are not included in the analysis. Comparing DNA migration patterns between small groups of smokers and nonsmokers indicated that long-term smoking had no effect on the subpopulation of cells that yield typical comets. Our results indicate that the SCGE assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human epithelial BM cells. (c) 2005 Wiley-Liss, Inc.

  2. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo


    The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a 60 Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 μm) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 μm) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 μm). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs

  3. The application of single cell gel electrophoresis or comet assay to human monitoring studies Aplicacion de la electroforesis unicelular o ensayo cometa en estudios de monitoreo humano

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    Mahara Valverde


    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.Objetivo. En la búsqueda de nuevos marcadores genotóxicos aplicables a estudios de poblaciones humanas expuestos a xenobióticos, la utilización del ensayo de electroforesis en una sola célula se ha propuesto como un método sensible y una buena alternativa. Material y métodos. Esta técnica detecta rompimientos en el ADN de cadena sencilla, así como sitios álcali lábiles y sitios retardados de reparación. Resultados. En este trabajo, presentamos nuestra experiencia utilizando este ensayo en poblaciones humanas expuestas ocupacionalmente o ambientalmente a diferentes xenobióticos. Conclusiones. Se discute la posible utilidad de este ensayo como un biomarcador de efecto genotóxico.

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

    African Journals Online (AJOL)

    The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to determine apoptosis induced by siRNA in Colo 320 cells. When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a ...

  5. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu


    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  6. In vitro mutagenicity and genotoxicity study of 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene, using the micronucleus test and the alkaline single cell gel electrophoresis technique (comet assay) in human lymphocytes. (United States)

    Tafazoli, M; Kirsch-Volders, M


    The main objective of this study was to compare the cytotoxic genotoxic and mutagenic activity of a number of chlorinated aliphatic hydrocarbons, which are widely used as chemical intermediates, solvents, degreasing agents etc. in industry, and to establish the structure-toxicity relationship of the chemicals by using the most adequate determinants in estimating their toxicity. The mutagenicity and cytotoxicity of some of the candidate chemicals, namely 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene were evaluated in an in vitro micronucleus assay. The cytokinesis-block methodology was applied on human lymphocytes in the presence or absence of an external metabolic activation system (S9-mix). In the micronucleus assay, all test substances, except 1,2,3-trichloropropane with and without S9-mix and 1,1,2-trichloroethane without S9-mix in the repeated experiment, exhibited a low but statistically significant mutagenic activity, compared to the concurrent control. However, none of the five chemicals was able to induce a clear and reproducible linear dose-dependent increase in micronucleus frequencies in this assay. Generally, mutagenic activity of the chemicals was found in the absence of severe cytotoxicity and/or cell cycle delay. The DNA breakage capacity and the cytotoxicity of these chemicals were also assessed in the alkaline single cell gel (SCG) electrophoresis test (comet assay) with and without S9-mix in isolated human lymphocytes. All chemical compounds induced DNA breakage, in the presence or absence of the metabolic activation system, at the doses tested. The data showed that the DNA reactivity of the chemicals increased with increasing degree of halogenation. The results of the present work suggested that the comet assay might be a more suitable and sensitive screening method than the micronucleus test for this particular class of compound. However, both assays do detect different

  7. Application of single cell gel electrophoresis in post-evaluation of organism radiation damage

    International Nuclear Information System (INIS)

    Jiang Lin; Mu Wanjun; Liu Guoping; Xu Yunshu; Luo Shunzhong; Gao Qingxiang


    The transient irradiation-caused DNA damage in the human peripheral blood lymphocytes,mouse peripheral blood lymphocytes and alive mouse irradiated by α-ray and γ-ray was investigated, and the single cell gel electrophoresis(SCGE, Comet Assay) was used to detect the extent of DNA damage. On this basis, the dose-effect curve and the evaluating method for radiant after-effect were established, the absorbed dose of alive mouse A irradiated by γ-rays was computed. The results indicate that not only the dose-effect can be described by using SCGE, but also the dose-computed after organism irradiated by radiant rays is achieved with it, and SCGE may be used as a new biological dosimeter. (authors)

  8. Application of single cell gel electrophoresis in post-evaluation of organism radiation damage

    International Nuclear Information System (INIS)

    Jiang Lin; Mu Wanjun; Liu Guoping; Xu Yunshu; Gao Qingxiang


    The immediate irradiation-caused DNA damage in the human Peripheral Blood Lymphocytes, mouse Peripheral Blood Lymphocytes and alive mouse irradiated by α-Rays and γ-rays was investigated, and the single cell gel electrophoresis(SCGE, Comet Assay) was used to detect the extent of DNA damage. On this base, the dose-effect curve and the evaluating method for radiant aftereffect were established, the absorbed does of alive mouse A irradiated by γ-rays was computed. The results indicated that not only the does-effect could be described by using SCGE, but also the does-computed after organism irradiated by radiant rays was achieved with it, and SCGE might be used as a new biological dosimeter. (authors)

  9. Investigation on accordance of DNA double-strand break of blood between in vivo and in vitro irradiation using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Liu Qiang; Jiang Enhai; Li Jin; Tang Weisheng; Wang Zhiquan; Zhao Yongcheng; Fan Feiyue


    Objective: To observe the consistency of DNA double-strand break between in vivo and in vitro irradiation, as a prophase study in radiation biodosimetry using single cell gel electrophoresis (SCGE). Methods: Detect DNA double-strand break after whole-body and in vitro radiation in mice lymphocytes using neutral single cell gel electrophoresis. The comet images were processed by CASP software and all the data were analysed by SPSS12.0. Results: There is no difference between in vivo and in vitro irradiation group in HDNA%, TDNA%, CL, TL, TM and OTM. Conclusion: The result of neutral single cell gel electrophoresis shortly after in vitro irradiation can precisely reflect the DNA double-strand break of lymphocytes in whole-body irradiation. (authors)

  10. Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji


    Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

  11. 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


    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)

  12. Determination of the Mutagenicity Potential of Supermint Herbal Medicine by Single Cell Gel Electrophoresis in Rat Hepatocytes

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    Zivar Amanpour


    Full Text Available Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes. Then cells were exposed to the Supermint herbal medicine at doses of 125, 250 and 500 μl/ml. Buffer 4 (incubation buffer and sodium dichromate were used as negative and positive control for one hour respectively. Then cell suspension with low melting point agarose were put on precoated slides and covered with agarose gel. Then lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. The parameter under this analysis was the type of migration which was determined according to Kobayashi pattern. Results: With increased dose of Supermint herbal medicine the DNA damage was slightly increased (P<0001. Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect.

  13. [Correlation of single-cell gel electrophoresis and mitomycin C-induced chromosomal breakage for chromosomal instabiligy in children with Fanconi anemia]. (United States)

    Zhang, Li; Liu, Qiang; Zou, Yao; Liu, Xiao-ming; Zhang, Jia-yuan; Wang, Shu-chun; Chen, Xiao-juan; Guo, Ye; Yang, Wen-yu; Ruan, Min; Liu, Tian-feng; Liu, Fang; Cai, Xiao-jin; Chen, Yu-mei; Zhu, Xiao-fan


    Fanconi anemia (FA) is characterized by bone marrow failure, congenital abnormalities and predisposition to neoplasia. Hypersensitivity of FA cells to the clastogenic effect of mitomycin C (MMC) provides a unique marker for the diagnosis before the beginning of hematological manifestations. The aim of this study was to evaluate the relationship between Single-Cell Gel Electrophoresis (SCGE) and mitomycin C-induced chromosomal breakage in children with FA. Between January 2007 and June 2011, 248 children (results of the two methods and compared with each other. The receiver operating characteristic (ROC) curve was used to evaluate the parameters in SCGE. Seventeen patients were diagnosed as FA and 231 as non-FA. Chromosomal breakage was found to be significantly higher in FA patients [(32.2 ± 4.8)%] than non-FA [(19.9 ± 3.0)%] and controls[(21.6 ± 4.8)%] when induced by MMC 80 ng/ml. The parameters of SCGE were significantly different between FA patients and non-FA or controls. All the parameters were rectilinearly correlated with MMC (P = 0.000). The most closely correlated parameter was the rate of comet cell (r = 0.848, P = 0.000). The results of ROC curves suggested the comet cell rate (0.999) was more important. SCGE might be used to discriminate between FA and non-FA individuals. The relationship between SCGE and MMC-induced chromosomal breakage was significant. The rate of comet cell was the important parameter.

  14. Improved single-cell gel electrophoresis assay for detecting DNA damage in Eisenia foetida. (United States)

    Di Marzio, Walter D; Saenz, María E; Lemière, Sebastien; Vasseur, Paule


    The earthworm (Eisenia foetida) is an attractive sentinel species for detecting genotoxicity in soil. In this study, an improved single-cell gel electrophoresis (SCGE) assay was developed for detecting DNA damage in the coelomocytes (lymphocytes) of earthworms. Coelomocytes were obtained from the coelomic fluid using a modified extrusion medium that did not include the mucolytic agent guaiacol. The extruded coelomocytes contained at least three types of cells: eleocytes (75% of the total), amoebocytes, and granulocytes. The DNA migration parameters were determined for untreated cells of each type in order that the assay could be performed with minimum inter- and intra-individual variation. In addition, lysis time was reduced to 10 min, and only one neutralization step was used. DNA damage was detected in isolated eleocytes treated with hydrogen peroxide and cadmium, and in eleocytes from earthworms exposed for up to 21 days to soil containing polycyclic aromatic hydrocarbons. The SCGE assay using earthworm eleocytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds.

  15. Preliminary study on biological dosimetry using alkaline single cell gel electrophoresis of human peripheral lymphocytes

    International Nuclear Information System (INIS)

    Liu Qingjie; Lu Xue; Feng Jiangbing; Chen Deqing; Chen Xiaosui


    Objective: To explore the feasibility of alkaline single cell gel electrophoresis (SCGE) in biological dosimetry of ionizing radiation. Methods: Normal peripheral blood samples from two healthy males were exposed to different doses coblat-60 gamma-rays, ranged from 0 to 5 Gy, and the tail length (TL) and Oliver tail moment (TM) of the lymphocytes were analyzed with SCGE. The dose-effect curves of TL and TM were fitted respectively. The TL and TM of lymphocytes for eight radiation workers were analyzed with SCGE, cumulative doses were estimated using the fitted TL and TM equations, and then compared with the recorded monitoring doses. Results: The TLs or TMs of normal human lymphocytes were increased with the irradiation doses, and its relationship can be fitted with a linear-quadratic equations: Y=13.59 + 20.87X - 2.27 X 2 for TL, and Y = 8.50 + 15.04X - 1.43X 2 for TM, respectively (Y denotes TL or TM value, X is radiation dose). The doses estimated with TM equation were closer to the recorded monitoring doses than that with TL equation. Conclusions: The TM in lymphocytes analyzed with SCGE is a promising radiation biological dosimeter. (authors)

  16. Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

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    Angelique eLewies


    Full Text Available The comet assay is a simple and cost effective technique, commonly used to analyse and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

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


    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. Comets

    International Nuclear Information System (INIS)

    Hughes, D.W.


    Comets are objects of considerable fascination and this paper reviews the present knowledge of the physical structure of the cometary nucleus, coma and tail, the orbits of comets in the Solar System, the proposed mechanisms of cometary origin, the decay processes suffered by comets, and the ways in which they can be observed from Earth and by spacecraft. (author)

  19. ASTRO Research Fellow Presentation - A comparison of the comet assay and pulsed-field gel electrophoresis as a predictive assay for radiosensitivity in human fibroblasts

    International Nuclear Information System (INIS)

    Sarkaria, Jann N.; Eady, John J.; Peacock, John H.; Steel, G. Gordon


    Purpose/Objective: To determine whether neutral lysis single-cell gel electrophoresis (comet assay) or pulsed-field gel electrophoresis (PFGE) can be used as a predictive assay for tissue response to radiotherapy as an alternative to clonogenic survival measurements. Materials and Methods: The comet assay has been widely used to measure DNA double strand breaks (dsb) in individual cells, and it has been suggested that it could be used as an alternative to clonogenic assays to measure radiosensitivity. Previous studies in this lab have demonstrated the ability of pulsed-field gel electrophoresis, which also measures DNA dsb, to accurately predict the radiosensitivity of a panel of fibroblasts based on determination of residual DNA dsb. As part of an ongoing study examining the relationship between fibroblast radiosensitivity and normal-tissue radiation reactions, we have compared the sensitivity and accuracy of the comet assay and PFGE on a different panel of non-transformed fibroblasts derived from breast cancer patients who developed severe radiation late effects and from case-matched controls. For the measurement of initial damage, cells were suspended in PBS and irradiated on ice for the comet assay and irradiated in agarose plugs on ice for pFGE. Residual damage was measured following irradiation of confluent cultures at 37 degree sign C and subsequent incubation for four hours prior to preparation of agarose slides and plugs. All irradiations were performed with a 59 TBq 60 Co source at a dose rate of 1.7 Gy/min. Electrophoresis was performed following neutral pH cell lysis. Comet images were captured and analyzed using Optimas software with DNA damage quantitated by the comet moment. PFGE gels were analyzed using a phosphor-image analysis system and damage was quantitated based on the percent of activity released from the well. Results: The comet assay was able to detect initial DNA damage at a threshold of 5 Gy and exhibited a linear dose

  20. The single cell gel electrophoresis: a potential tool for biological dosimetry

    International Nuclear Information System (INIS)

    Bhilwade, H.N.; Chaubey, R.C.; Rajagopalan, Rema


    In this paper, observations made on the effect of gamma radiation on DNA strand breaks in human leucocytes using alkaline comet assay are communicated. Human peripheral blood was collected from healthy volunteers and exposed in vitro to different doses of gamma radiation in the medium 0.125, 0.25, 0.50 and 1.0 Gy and high doses range of 2.04.0 and 8 Gy, using a 6 0C o Teletherapy machine at a dose rate of 0.668 Gy/min at 0 deg C in air. Migration of DNA fragments (TL) and tail moment (TM) was taken as the criteria of genetic damage and measured using the application software, SCGE-Pro, developed in our laboratory. Data were analyzed using one-way ANOVA for statistical significance. A dose dependent increase in the TL (p<0.001 ) and TM (p<0.001 ) was observed for DNA single strand breaks in the medium dose range, from 0.125 to 1.00 Gy dose. Similarly, data on DNA strand breaks from the high dose (e.g. 2 to 8 Gy) experiments also showed a significant increase in TL (p<0.001) and TM (p<0.001) at all the doses tested. The major finding of these experiments was the detection of DNA single strand breaks in human leucocytes, even at the lowest dose of 0.125 Gy. (author)

  1. Evaluation of genotoxicity after application of Listerine(R) on human lymphocytes by micronucleus and single cell gel electrophoresis assays. (United States)

    Türkez, Hasan; Togar, Basak; Arabaci, Taner


    Listerine (LN) is one of the most commonly used mouth rinses worldwide although very limited information is available concerning its genotoxicity. In another view, the biological safety profile of oral care products is frequently assumed on the basis of simplistic test models. Therefore, the present study was undertaken to investigate the in vitro genotoxic potential of LN using micronucleus and single cell gel electrophoresis tests as genetic endpoints. Different concentrations of LN (0-100% of ml/culture, v/v) were applied to whole human blood cultures (n = 5). The result of the present study showed that there were no statistically significant differences (p > 0.05) between the control group and the groups treated with LN alone in both analysed endpoints. In conclusion, our result first demonstrated the absence of genotoxicity of LN on human lymphocytes.

  2. Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt-tungsten carbide. (United States)

    Van Goethem, F; Lison, D; Kirsch-Volders, M


    Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand and/or double strand breaks in a cell by cell approach, it is new possible to address this question at the cellular level. We therefore compared the genotoxic potential of pure cobalt powder (Co) and a cobalt-containing alloy, cobalt-tungsten carbide (WC-Co), involved in specific lung disorders, in parallel with the alkaline single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (MN) test, both carried out in vitro on isolated human leukocytes. The comet assay indicated that the WC-Co mixture produced a higher level of DNA damage than Co alone; WC alone was not able to induce a dose-dependent DNA breakage effect as was seen for Co and WC-Co. Results from the MN test confirmed these observations. It was clear that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC and suggested that their physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers. In agreement with data obtained in the same laboratory on liposoluble chemicals (PCBs and chlorinated aliphatic hydrocarbons) and from the literature, the results indicate that both the comet assay and the micronucleus test were able to detect differences in the genotoxic potential of the compounds studied. Although the micronucleus test seemed to be less sensitive to assess a synergistic DNA damaging potential of the mixture involved, it detects chromosomal aberrations (chromosome/genome mutations

  3. Mutagenicity assessment of two herbal medicines, Urtan and Carmint in human leukocytes by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Jalili, M.; Hatami, A.; Kalantari, H.; Kalantar, E.


    The use of herbal medicine is an old and still widespread particle, which makes studies their genotoxicity essential. Urtan and Carmint are examples of herbal medicines used in Iran which used for the treatment of hyperplasia, diuretic, urinary diffusion and antispasmodic action, carminative gastrointestinal disfunction respectively. The aim of this study was to evaluate the mutagenicity of these herbal medicines in human leukocytes by Single Cell Gel Electrophoresis (SCGE). Both the herbal medicines were used at four concentrations (0.046, 0.092, 0.138 and 0.184/ 100 ml). The results were compared against positive (Cr VI) and negative (no mutagen) control groups. Fluorescence microscope was used to observe the DNA damage in randomly selected cells, which were stained with ethidium bromide. Microscopic observation of the affected cells due to Urtan and Carmint was encouraging as compared to previous studies using SCGE. Both Urtan and Carmint exhibited considerable DNA damage to the blood cells. For example, Urtan at o.184 g/100ml concentration had almost 23% mutagenic effect and as the concentration increased the mutagenic effect also increased. Similarly Carmint exhibited considerable DNA damage on blood cells. Therefore, both the herbal medicines may have some mutagenic effect. (author)

  4. Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells

    International Nuclear Information System (INIS)

    Gedik, C.M.; Collins, A.R.; Ewen, S.W.B.


    The authors have adapted procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m -2 and incubation for as little as 5 min. The authors also studied UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, key incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examined consequent breaks both by SCGE and by alkaline unwinding to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10 9 daltons are detected. (Author)

  5. Use of single cell gel electrophoresis assays for the detection of DNA-protective effects of dietary factors in humans: recent results and trends. (United States)

    Hoelzl, Christine; Knasmüller, Siegfried; Misík, Miroslav; Collins, Andrew; Dusinská, Maria; Nersesyan, Armen


    This article summarises the results of human dietary intervention trials employing the comet assay (single cell gel electrophoresis, SCGE), which have been published in the last few years (i.e., between 2005 and 2008) and describes new trends and developments as well as current problems concerning the design of intervention trials and the interpretation of the results. Most new studies were carried out with complex plant derived foods and juices; only a few were conducted with individual food constituents. With specific vegetables, for example with water cress and Brussels sprouts, potent antioxidant effects were observed; also coffee caused a protective effect and it is notable that it was more effective than consumption of a diet containing increased levels of fruits and vegetables. Interesting recent developments include the development of protocols which enable us to monitor protection towards genotoxic chemicals contained in the human diet, and it was shown in preliminary studies that alterations of the activities of drug metabolising enzymes by dietary factors lead to altered sensitivity of lymphocytes against DNA damage caused by certain dietary carcinogens. Another novel approach is the development of methods to monitor the effects of dietary factors on DNA repair. The development of protocols for experiments with exfoliated buccal cells is another potentially valuable innovation. The adequate experimental design of SCGE trials is still a matter of debate and the evaluation of the available data shows that there is an urgent need to develop guidelines concerning the number of participants, sampling periods, duration of trials, use of placebos, and definition of adequate run-in and wash-out phases. Recent studies showed that the results of dietary studies could be biased by factors such as age, sex, body mass index and life style habits and by seasonal effects. Another still unsolved problem is the interpretation of the results of SCGE trials in regard to

  6. Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.

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    Satoru Kaneko

    Full Text Available Single-cell pulsed-field gel electrophoresis (SCPFGE with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

  7. In vitro genotoxic effects of hard metal particles assessed by alkaline single cell gel and elution assays. (United States)

    Anard, D; Kirsch-Volders, M; Elhajouji, A; Belpaeme, K; Lison, D


    Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers.

  8. Validation of comet assay using different genotoxicity tests in mammals

    International Nuclear Information System (INIS)

    Balid, Arundhati; Godbole, N.N.; Bhilwade, H.N.; Rajagopalan, R.; Chaubey, R.C.


    Present studies were undertaken to compare the sensitivity of three test systems: chromosome aberration test, micronucleus test and single cell gel electrophoresis/comet assay. These tests were carried out in a single animal in order to optimize, evaluate and compare their relative sensitivities

  9. Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Sanjit Mukherjee


    Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA. Pearson′s product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe′s pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD tail lengths (in mm for cancer (24.95 ± 5.09 and leukoplakia (12.96 ± 2.68 were significantly greater than in controls (8.54 ± 2.55, P<0.05. After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92, the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77 was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001. Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

  10. Detection of DNA damage by comet fluorescence in situ hybridization. (United States)

    Schlörmann, Wiebke; Glei, Michael


    Comet fluorescence in situ hybridization (Comet-FISH) is a useful method to detect overall and region-specific DNA damage in individual cells. Two well-established methods are combined, the Comet assay (single cell gel electrophoresis) and fluorescence in situ hybridization (FISH). The Comet assay is the method of choice for the detection of DNA damage. With the alkaline version the influence of specific substances such as water pollutants or ingredients of food on individual cells can be easily measured. The Comet assay involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. Damaged DNA migrates from the nucleus (head of the comet) forming a tail. The percentage of DNA in the tail correlates with the degree of DNA strand breaks (DNA damage). The combination of FISH with the Comet assay uses labeled probes which hybridize specifically to selected DNA sequences. This allows the detection of specific DNA damage or repair capacity in single cells. Here we present exemplarily the Comet-FISH method by detection of DNA damage using hydrogen peroxide as a genotoxic model substrate.


    Investigation ofDNA Repair by Sister Chromatid Exchange (SCE) Analysis and the Alkaline Single Cell Gel Assay (SCG) in Mammalian Go-Lymphocytes after In Vitro Exposure to Ethylene Oxide (EO). EO is a large volume chemical used primarily as an intermediate in manufacturing...

  12. Protective effects of Brussels sprouts towards B[a]P-induced DNA damage: a model study with the single-cell gel electrophpresis (SCGE)/Hep G2 assay

    NARCIS (Netherlands)

    Laky, B.; Knasmuller, S.; Gminski, R.; Mersch-Sundermann, V.; Scharf, G.; Verkerk, R.; Freywald, C.; Uhl, M.; Kassie, F.


    The aim of this study was to investigate the chemoprotective effects of Brussels sprouts juice towards benzo[a]pyrene (B(a)P)-induced DNA damage in the single-cell gel electrophoresis (SCGE)/Hep G2 test system. This assay combines the advantages of the SCGE assay with that of the use of

  13. Assessment of vinyl chloride-induced DNA damage in lymphocytes of plastic industry workers using a single-cell gel electrophoresis technique. (United States)

    Awara, W M; El-Nabi, S H; El-Gohary, M


    DNA damage and the formation of stable carcinogen-DNA adducts are considered critical events in the initiation of the carcinogenic process. This study was carried out to assess whether exposure of plastics industry workers to the vinyl chloride monomer (VCM) for different periods of time would cause DNA damage, using the single-cell gel electrophoresis (SCGE) technique. Levels of DNA damage was assessed by both extent of DNA migration and numbers of DNA damaged spots in the peripheral blood lymphocytes from 32 plastics workers with different periods of exposure to VCM; they were evaluated by comparison with a group of non-exposed individuals. It was found that plastics workers who were exposed to VCM for different periods of time showed significantly increased levels of DNA damage compared with the non-exposed subjects. There was a significant correlation between the severity of DNA damage and duration of exposure. However, no significant correlation was found between the age of all subjects and DNA damage. Concentrations of VCM in the air inside the factory were found to be significantly higher than values in non-exposed areas, despite being lower than the threshold limit value (TLV). Our results encourage the application of SCGE as a sensitive, simple, fast and useful technique in the regular health screening of workers occupationally exposed to VCM (even at concentrations below the TLV) to assess the possibility of any DNA damage.

  14. An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

    DEFF Research Database (Denmark)

    Johansson, Clara; Møller, Peter; Forchhammer, Lykke


    The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to dif...

  15. Kinetics of DNA strand breaks and protection by antioxidants in UVA- or UVB-irradiated HaCaT keratinocytes using the single cell gel electrophoresis assay. (United States)

    Lehmann, J; Pollet, D; Peker, S; Steinkraus, V; Hoppe, U


    The aim of this study was to characterize the genotoxic action of UVA and UVB in human keratinocytes by application of the single cell gel electrophoresis assay (SCGE assay). Dose dependence of DNA damage, the time course of its repair, and the influence of cellular antioxidant status were assessed. Irradiation with UVA or UVB both resulted in a dose-dependent increase in the level of DNA damage. A time course study to evaluate the repair kinetics in keratinocytes irradiated with 5 J/cm2 UVA revealed an immediate occurrence of DNA effects which subsequently disappeared within about 1 h, indicating removal of DNA lesions. This rapid repair of DNA damage is consistent with the observation that 5 J/cm2 UVA did not impair cellular viability. In contrast, exposure to 15 mJ/cm2 UVB resulted in a prolonged repair of DNA damage which lasted about 25 h. Thus, the repair kinetics of UVA- and UVB-induced DNA damage clearly differed from each other, implicating the induction of different types of DNA lesions by UVA and UVB. Neither a pretreatment with Mg-ascorbyl phosphate or D,L-alpha-tocopherol, nor depletion of endogenous glutathione altered cellular sensitivity to UVB. In contrast, the DNA damaging effects of UVA could be counteracted by a pretreatment with these antioxidants. These observations confirm that the UVA-induced effects on DNA are related to radical mediated strand breaks and DNA lesions forming alkali-labile sites. The UVB-induced effects mainly occur as a consequence of excision repair-related strand breaks. The observed repair kinetics of DNA lesions and the influence of cellular antioxidant status may help to elucidate protective mechanisms against the carcinogenic effects of UV radiation present in sunlight.

  16. Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

    DEFF Research Database (Denmark)

    Møller, Peter; Möller, Lennart; Godschalk, Roger W L


    The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

  17. Genotoxicity effects of nano bioactive glass and Novabone bioglass on gingival fibroblasts using single cell gel electrophoresis (comet assay: An in vitro study

    Directory of Open Access Journals (Sweden)

    Mohammad Tavakoli


    Conclusion: The findings of this study have demonstrated that novel nano bioactive glass had no genotoxicity in concentrations lower than 4 mg/ml. Nanoparticles have a higher surface area in comparison to microparticles and thus, the amount and rate of ion release for nanoparticles are extremely higher. This difference is the main reason for the different genotoxicity of nano bioactive glass and micro Novabone bioglass in the concentrations higher than 4 mg/ml.

  18. Single cell gel electrophoresis as a tool to assess genetic damage in Heleobia cf. australis (Mollusca: Gastropoda as sentinel for industrial and domestic pollution in Montevideo bay (Uruguay

    Directory of Open Access Journals (Sweden)

    Silvia Villar


    Full Text Available AbstractThe knowledge of the extent of DNA damage in aquatic organisms in polluted areas is an important issue because contamination may alter their health at sublethal levels. Although molluscs have been widely used to monitor water pollution, there are no records of in vivo genotoxicity studies. Heleobia cf. australis, is distributed in almost all Uruguayan coastal ecosystems, including highly polluted sites. The comet assay is a damage genetic biomarker based on the migration of negatively charged DNA fragments produced by mutagenic agents in individual cells. Live individuals were collected in the Montevideo Bay (impacted area and Laguna Garzón (control to analyze the presence of mutagenic agents in the former site through comet assay. Cells from organisms of the impacted area showed significantly higher levels of genetic damage than those obtained in the control population, measured by percentage of DNA in the tail. Although preliminary, this approach supports the idea that H. cf. australis could be used as a sentinel to evaluate the presence of mutagenic agents in estuarine environments, alerting to the impact of contamination in its early stages.

  19. Single-Cell Western Blotting. (United States)

    Sinkala, Elly; Herr, Amy E


    Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

  20. DNA-electrophoresis of single cells - a method to screen for irradiated foodstuffs

    International Nuclear Information System (INIS)

    Leffke, A.; Helle, N.; Boegl, K.W.; Schreiber, G.A.


    Microelectrophoresis of single cells can be used to detect γ-irradiation over a wide dose range and for a variety of products. It is a simple and rapid test for DNA damages and can be used for screening. The method was tested on cell suspensions of bone marrow and muscle cells from frozen chicken legs, chicken heart, turkey liver, beef and pork irradiated with doses up to 3 kGy. Cell suspensions were prepared by incubation of tissues in EDTA-SDS-buffer at pH 8. Single cell electrophoresis was performed in 0.75% agarose gel. DNA was visualised by silver staining. In unirradiated samples no or only a small amount of DNA penetrated the cell membranes. Cells of irradiated samples appeared like a ''comet'' due to to migration of DNA-fragments out of cell. (orig.)

  1. Assessment of DNA damage and Chromosome aberration in human lymphocyte exposed to low dose radiation detected by FISH(Fluorescence In Situ Hybridization) and SCGE(Single Cell Gel Electrophoresis)

    International Nuclear Information System (INIS)

    Chung, Hai Won; Kim, Su Young; Kim, Byung Mo; Kim, Sun Jin; Ha, Sung Whan; Kim, Tae Hwan; Cho, Chul Koo


    Comparative study was performed for the assessment of DNA damage and Chromosomal aberration in human lymphocyte exposed to low dose radiation using Fluorescence In Situ Hybridization(FISH) and Single Cell Gel Electrophoresis(SCGE). Chromosomal aberrations in human lymphocyte exposed to radiation at doses of 5, 10, 30 and 50cGy were analysed with whole chromosome-specific probes by human chromosome 1, 2 and 4 according to PAINT system. FISH with chromosome-specific probe has been used to be a valid and rapid method for detection of chromosome rearrangements induced by low dose radiation. The frequencies of stable translocation per cell equivalents were 0.0116, 0.0375, 0.0407, 0.0727 and 0.0814 for 0, 5, 10, 30 and 50cGy, respectively, and those of dicentric were 0.00, 0.0125, 0.174, 0.0291 and 0.0407 respectively. Radiation induced DNA damage in human lymphocyte in a dose-dependent manner at low doses from 5cGy to 50cGy, which were analysed by single Cell Gel Electrophoresis(SCGE). From above results, FISH seemed to be useful for radiation biodosimetry by which the frequencies of stable aberrations in human lymphocyte can be observed more easily than by conventional method and SCGE also seemed to be sensitive method for detecting DNA damage by low dose radiation exposure, so that those methods will improve our technique to perform meaningful biodosimetry for radiation at low doses

  2. The pursuit of the genome instability by comet assay (single cell gel electrophoresis) in patients with cancer of the cavum in western Algerian; La recherche de l'instabilite genomique par le test des cometes (single cell gel electrophoresis) chez les malades atteints d'un cancer du cavum dans l'Ouest algerien

    Energy Technology Data Exchange (ETDEWEB)

    Boukerche, A.; Dali-Youcef, A.F. [Service de Radiotherapie, Oran (Algeria); Bouali-Youcef, Y. [Laboratoire d' Immunologie, Oran (Algeria); Mehadji, M. [Service d' ORL, Oran (Algeria); Chenal, C. [Rennes-1 Univ., UMR CNRS 6853, 35 (France)


    The analysis of results has shown a constitutional genome instability among the patients with a cavum cancer with a defect in DNA repair where some exogenous factors ( Epstein-Barr virus, EBV) seem play an important part. (N.C.)

  3. Single cell-resolution western blotting. (United States)

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E


    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

  4. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay. (United States)

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine


    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

  5. Application of the comet assay in studies of programmed cell death (PCD in plants

    Directory of Open Access Journals (Sweden)

    Maria Charzyńska


    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.

  6. Observing comets

    CERN Document Server

    James, Nick


    Since comet Shoemaker-Levy collided with the planet Jupiter with stupendous force in 1994 there has been an upsurge of amateur interest in comets Most comets are first discovered by amateur astronomers because there are so many amateurs looking for them, and techniques and instruments have improved dramatically in the past few years After a short but detailed introduction to the comets themselves Nick James and Gerald North describe comet hunting, photographing and imaging comets, and digital image processing The use of computers for orbital calculations and even helping to discover new comets is given a full chapter, as are advanced techniques including comet photometry and spectroscopy This comprehensive book has an accompanying CD-ROM and is at once a "primer" for comet hunters and a reference text for more advanced amateur astronomers

  7. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification (United States)

    Ge, Jing; Chow, Danielle N.; Fessler, Jessica L.; Weingeist, David M.; Wood, David K.; Engelward, Bevin P.


    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies. PMID:25527723

  8. Assessment of Genotoxicity of Ionizing radiation using Tradescantia-Comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Ryu, Tae Ho; Hyun, Kyung Man; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Wilhelmova, Nad [Institute of Experimental Botany, Prague (Czech Republic)


    Over the last two decades, several new methodologies for the detection of DNA damage have been developed. The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, also called the single cell gel electrophoresis (SCGE) was first introduced by Ostling and Johanson as a microelectrophoretic technique for the direct visualization of DNA damage in individual cells. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Although the genotoxic effects detected by Tradescantia tests cannot be associated with mutagenesis or even carcinogenesis in humans, these bioassays are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay

  9. Great Comets (United States)

    Burnham, Robert


    Spectacular and mysterious objects that come and go in the night sky, comets have dwelt in our popular culture for untold ages. As remnants from the formation of the Solar system, they are objects of key scientific research and space missions. As one of nature's most potent and dramatic dangers, they pose a threat to our safety--and yet they were the origin of our oceans and perhaps even life itself. This beautifully illustrated book tells the story of the biggest and most awe-inspiring of all comets: those that have earned the title "Great." Robert Burnham focuses on the Great comets Hyakutake in 1996 and Hale-Bopp in 1997, which gripped attention worldwide because, for many, they were the first comets ever seen. He places these two recent comets in the context of their predecessors from past ages, among them the famous Comet Halley. Great Comets explains the exciting new discoveries that have come from these magnificent objects and profiles the spaceprobes to comets due for launch in the next few years. The book even takes a peek behind Hollywood's science-fiction fantasies to assess the real risks humanity faces from potential impacts of both comets and asteroids. For everyone interested in astronomy, this exciting book reveals the secrets of the Great Comets and provides essential tools for keeping up to date with comet discoveries in the future. Robert Burnham has been an amateur astronomer since the mid-1950s. He has been a senior editor of Astronomy magazine (1986-88) and is the author of many books and CD-ROMS, including Comet Hale-Bopp: Find and Enjoy the Great Comet and Comet Explorer.

  10. Application of the DNA ''comet assay'' to detect irradiation treatment of foods

    International Nuclear Information System (INIS)

    Delincee, H.


    Since treatment with ionising radiation causes DNA fragmentation, microgel electrophoresis of single cells (''comet assay'') offers a simple and rapid tool for identification of irradiated foods. The principle is based on migration of DNA in an agarose gel exposed to an electric field. Single cells or nuclei are embedded in the agarose and after lysis, intact DNA will virtually not move out of the cell upon electrophoresis, whereas if DNA has been fragmented, the fragments are able to migrate and ''comets'' following the cells become visible after staining. The advantages of this test, however, for foods not exposed to heat are its speed and simplicity, as the electrophoretic separation only requires a few minutes. DNA is visualised by silver staining, avoiding the need for a fluorescence microscope. Thus the method requires only relatively cheap equipment - in contrast to other methods for identification of irradiated food such as electron spin resonance (ESR) spectroscopy or gas chromatography/mass spectrometry (GC/MS). The DNA ''comet assay'' , therefore, seems suitable as a pre-screening test to detect whether food has been radiation processed. Suspected samples may subsequently be analysed by established, but more expensive techniques. (author)

  11. Localized irradiations, evaluation through 'Comet Assay'

    International Nuclear Information System (INIS)

    Di Giorgio, Marina; Taja, Maria R.; Nasazzi, Nora B.; Bustos, N.; Cavalieri, H.; Bolgiani, A.


    During the last 50 years various radiation accidents involving localized irradiations occurred, resulting mainly from improper handling of sealed sources of Cobalt 60, Cesium 137 or Iridium 192 at work placed for industrial gammagraphy and other radiation sources. Severe skin reaction may developed at the contact sites. Such inhomogeneous irradiations lead to a differential exposure of lymphocytes in lymphatic tissues or other organs that may recirculate into the peripheral blood producing a mixed irradiated and unirradiated population of lymphocytes. Applying the mathematical models 'Contaminated Poisson' of Dolphin and Qdr method of Sasaki, a mean dose in the irradiated body area and its size can be estimated from unstable chromosome aberration scoring. There are also different biophysical techniques that can give response in localized irradiations. Biological dosimetry is a necessary complement to physical and clinical dosimetries. Thus, there is increasing interest in the assessment of biological markers that permit the detection of radiation induced damage in the localized irradiations. The 'Comet Assay' (single cell gel electrophoresis) is a sensitive, rapid and relatively inexpensive method for measuring DNA damage in individual cells. Single cells are embedded in agarose on microscope slides, lysed to remove the majority of the proteins, electrophoresed, then stained with ethidium bromide in order to visualize the DNA. When visualized using a fluorescent microscope, DNA of undamaged cells appears as a spherical mass occupying the cavity formed by the lysed cell. Following radiation damage, the smaller the fragment size and the grater the number of fragments of DNA, the grater the percentage of DNA that it is able to migrate in an electric field, forming a comet image. The assay can be performed under alkaline conditions to examine DNA single strand breaks (SSBs), or in non denaturing (neutral) conditions to measure double strand breaks (DSBs) in individual

  12. Protective effect of peach kernel extracts on radiation-induced DNA damage in human blood lymphocytes in the comet assay

    International Nuclear Information System (INIS)

    Kim, Jin Kyu; Lee, Chang Joo; Park, Tae Won; Chai, Young Gyu


    The alkaline single-cell gel electrophoresis (SCGE) assay, the comet assay, has been applied to the detection of DNA damage from a number of chemical and biological factors in vivo and in vitro. The comet assay is a novel method to assess DNA single-strand breaks, alkali-labile sites in individual cells. We evaluated the effect of peach kernel extracts on radiation-induced DNA damage in human blood lymphocytes using the comet assay. The lymphocytes, with or without pretreatment of the extracts, were exposed to 0, 0.1, 0.3, 0.5, 1.0 and 2.0 Gy of 60 Co gamma ray. Significantly increased tail moment, which was a marker of DNA strand breaks in the comet assay, showed an excellent dose-response relationship. The treatment of the peach kernel extracts prominently reduced the DNA damage in irradiated groups compared to that in non-treated control groups. The result indicated that the extracts showed radioprotective effect on lymphocyte DNA when assessed by the comet assay

  13. Benzene-induced genotoxicity in mice in vivo detected by the alkaline comet assay

    DEFF Research Database (Denmark)

    Tuo, J; Loft, S; Thomsen, M S


    The myelotoxic and genotoxic effects of benzene have been related to oxidative DNA damage after metabolism by CYP2E1. Single cell gel electrophoresis (alkaline comet assay) detects DNA damage and may thus be a convenient method for the study of benzene genotoxicity. Benzene exposure to NMRI mice...... as a single oral gavage at 40, 200 or 450 mg/kg resulted in dose-related DNA damage indicated by an increased comet tail length of peripheral blood lymphocytes and bone marrow nucleated cells sampled 6 h after exposure. After a dose of 40 mg/kg, there was a 1.6-fold increase of 'tail length' in bone marrow.......01). By comparing our data with those from genotoxicity studies on benzene using other methods, we conclude that the 'alkaline comet assay' is a sensitive method to detect DNA damage induced by benzene. We also infer that CYP2E1 contributes, at least partly, to the formation of the 'comet'-inducing metabolites...

  14. 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...

  15. Comet assay. Pt.1. Theory and practice

    International Nuclear Information System (INIS)

    Kruszewski, M.; Wojewodzka, M.; Iwanenko, T.


    Comet assay is a new method for measuring DNA breakage in a single cell. The main applications of the method are estimation of DNA single and double strand breaks, oxidative damage, pyrimidine dimers and (6-4)photoproducts, DNA-DNA and DNA-protein crosslinks. The method is used for studying DNA damage and its repair. (author).19 refs, 9 figs

  16. OpenComet: An automated tool for comet assay image analysis

    Directory of Open Access Journals (Sweden)

    Benjamin M. Gyori


    Full Text Available Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

  17. Comet assay for rapid detection of base damage in foods

    International Nuclear Information System (INIS)

    Al-Zubaidi, I. A.; Abdullah, T. S.; Qasim, S. R.


    Single cell gel electrophoresis (SCGE) or comet assay technique a sensitive, reliable and rapid method for DNA double and single strand break, alkali- labile site and delayed repair site detection in individual cells. In recent years, this method has been widely used for studies of DNA repair, genetic toxicology, and environmental biomontoring, however, this technique serves as an important tool for detection of DNA damage in living organism and is increasing being used in genetic testing of industrial chemicals, environmental agent's contaminations. This research paper helps to evaluate the oxidant agent's effects of exposure to organic pollutants by using comet assay techniques. This study used five samples of each food sample (Meat, Chicken, Rice, Fruits, Vegetables and Tea) to evaluate the genotoxic effects of exposure, to environmental agent's pollutants. The experimental data suggest that the DNA damage parameters ( Tail length, Tail width 1 ) were found higher value in exposed population when compared with the ratio of the length to width that cells exhibiting no migration having a ratio of 1. The percentage and distribution of cells in exposed population of cells also increases with the increase in values. This study demonstrates that, using sensitive techniques, it is possible to detect environmental agent's risks at an early stage. (Author)

  18. The JaCVAM / OECD activities on the comet assay

    Directory of Open Access Journals (Sweden)

    Hajime Kojima


    Full Text Available The in vivo alkaline single cell gel electrophoresis assay, also called alkaline comet assay is a method measuring DNA strand breaks in eukaryotic cells. This assay was adopted in the Organisation for Economic Co-operation and Development (OECD Test guideline (TG 489 on September 26, 2014. This TG is part of a series of TGs on genetic toxicology. A formal validation trial of the this assay was performed in 2006-2012, coordinated by the Japanese Center for the Validation of Alternative Methods (JaCVAM, in conjunction with the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM and the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM . The assay was reviewed by the OECD genotoxicity experts based on the JaCVAM trial (2014 and in Rothfuss et al. (2010. This TG includes the recommended use and limitations of the comet assay, and is based on the final protocol used in the validation trial, and on additional relevant published and unpublished (laboratories proprietary data. The outline of this TG describes below: each treated group is composed of a minimum of 5 animals of one sex (or of each sex as appropriate. A positive and a vehicle control group are also used. Administration of the treatment consists of daily doses over duration of 2 days or more, ensuring the test chemical reaches the target tissue which can be the liver, the kidney or other tissues if justified. Tissues of interest are dissected and single cells/nuclei suspensions are prepared and embedded in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis at high pH. This results in structures resembling comets which by using suitable fluorescent stain, can be observed by fluorescent microscopy. Based on their size

  19. Whence comets? (United States)

    A'Hearn, Michael F


    Recent advances in cometary science have indicated the importance of mixing of materials in the disk where the planets of our solar system formed. Now, the results from the Stardust Discovery Mission unambiguously show that even more extensive and earlier mixing of the material took place, raising new challenges for theories of the protoplanetary disk and the formation of comets.

  20. Single cell electroporation on chip

    NARCIS (Netherlands)

    Valero, Ana


    In this thesis the results of the development of microfluidic cell trap devices for single cell electroporation are described, which are to be used for gene transfection. The performance of two types of Lab-on-a-Chip trapping devices was tested using beads and cells, whereas the functionality for

  1. Single Cell Isolation and Analysis

    Directory of Open Access Journals (Sweden)

    Ping Hu


    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.

  2. Assessment of radiation induced cytogenetic damage in human keratinocytes by comet assay

    International Nuclear Information System (INIS)

    Joseph, Praveen; Sanjeev Ganesh; Narayana, Y.; Puthali, Abhay; Bhat, N.N.


    In the present study the effect of gamma radiation on normal human keratinocytes (HaCaT) cells has been analyzed using alkaline comet assay and a comparative study over the sensitivity of different comet parameters such as tail length (TL), olive tail moment (OTM) and percentage tail DNA (TDNA) has also been made. Human keratinocytes (HaCaT) cells were grown in Dulbecco's modified essential medium (DMEM) (10% FCS) at 37 °C in a humidified atmosphere containing 5% CO 2 . Cultured cells were harvested with 0.025 % trypsin EDTA. The sample (2 X 10 cells/ml) was exposed to gamma radiation of different dose using a 60 Co gamma source at dose rate of 2 Gy min -1 and the dosimetry has been carried out using Fricke and FBX dosimeters. After irradiation, to quantify the DNA damage the comet assay (single cell gel electrophoresis) was carried out under alkaline conditions, by the methods outlined by Singh et al. The quantification of the DNA strand breaks in each cells were performed using CASP software. The DNA damage quantification can be accomplished by measuring those comet parameters which exhibit a linear dependence on the amount of DNA damage. In the present study, comet parameters such as OTM, TL and TDNA were recorded and the variation of these parameters and their correlation coefficients for different doses of gamma radiation is plotted. The OTM value is normalized with control value and control for TL and TDNA is adjusted to zero to avoid initial variations in different experiments

  3. Plant single-cell and single-cell-type metabolomics. (United States)

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue


    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Assessment of electron beam-induced DNA damage in larvae of chestnut weevil, Curculio sikkimensis (Heller) (Coleoptera: Curculionidae) using comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Todoriki, Setsuko [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan)]. E-mail:; Hasan, Mahbub [Laboratory for Stored Product Protection, Department of Zoology, Rajshahi University, Rajshahi 6205 (Bangladesh); Miyanoshita, Akihiro [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan); Imamura, Taro [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan); Hayashi, Toru [Radiation and Information Technology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642 (Japan)


    Effect of electron beam treatment on DNA damage in mature larvae of chestnut weevil Curculio sikkimensis (Heller) was assessed using single-cell gel electrophoresis (DNA comet assay). Electrons at acceleration voltages of 0 (control), 300, 750, 1000, and 1500 kV at radiation doses of 1 and 4 kGy were used. Electron beam-treated chestnut larvae showed typical DNA fragmentation, compared with cells from non-treated ones which showed a more intact DNA. Investigations using the comet assay showed that the parameters including tail length, tail moment, olive tail moment as well as the quota of DNA damage at both the doses were significantly larger than the control batch larvae. Thus, this technique could contribute to analytical identification of an effective disinfestation and quarantine treatment.

  5. Single-cell western blotting. (United States)

    Quadri, Syed M S


    Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

  6. DNA-repair measurements by use of the modified comet assay

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia


    The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating...... laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell...

  7. Single-cell photoacoustic thermometry. (United States)

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


    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.

  8. Measuring single-cell density. (United States)

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


    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.

  9. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

    Directory of Open Access Journals (Sweden)

    Adriana Díaz


    Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

  10. Hemocytes of zebra mussels (Dreissena polymorpha are relevant cells for the monitoring of environmental genotoxicity by the comet assay.

    Directory of Open Access Journals (Sweden)

    Marc Bonnard


    Full Text Available The measure of DNA integrity by the single cell gel electrophoresis (SCGE or comet assay is especially recommended for its sensitivity and its capacity for detecting different types of damages. Therefore, it has been applied in environmental genotoxicity in a variety of organisms. It appears today necessary to define both reference and threshold levels of DNA damage, for their application in in situ biomonitoring. However, little is known about the influence of both biological (sex, reproduction status or external (temperature… confounding factors on the measure of DNA damage by the comet assay. These variables need to be taken into account if the robustness of the assay is to be established (Jha, 2008. In the zebra mussel Dreissena polymorpha (recommended as a sentinel species in the evaluation of freshwater quality the measure of DNA damage by the comet assay is mainly performed on hemocytes, which are circulating cells involved in key physiological functions such as immunity, homeostasis, detoxication…. This communication will present and discuss results from an innovative study about the variability of the baseline level of DNA damage in hemocytes of mussels encaged for one year in the canal de l’Aisne à la Marne (Reims, according to their sex and their reproductive status. The sensitivity and the suitability of hemocytes in the evaluation of environmental genotoxicity will also be discussed, referring to observations during a 6 month-exposure of mussels in mesocosms to environmentally realistic concentrations of carbamazepine.

  11. Single-Cell Genomic Analysis in Plants

    Directory of Open Access Journals (Sweden)

    Yuxuan Yuan


    Full Text Available Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis.

  12. DNA damage in birds after the mining waste spill in southwestern Spain: a Comet assay evaluation. (United States)

    Pastor, N; López-Lázaro, M; Tella, J L; Baos, R; Forrero, M G; Hiraldo, F; Cortés, F


    In April 1998, an ecological disaster resulting from a massive toxic spill of mining acid waste rich in heavy metals posed a serious threat to the Doñana National Park in southwestern Spain. This especially important protected area is the nesting and breeding site for many endangered bird species; white storks (Ciconia ciconia) and black kites (Milvus migrans) are considered the more representative. The suitability of the Comet assay as a biomarker for genotoxic analysis in environmental biomonitoring has been recently validated in studies using different sentinel organisms such as fish, amphibians, rodents, or mollusks. Birds preying on a variety of invertebrate and vertebrate species in the marshlands are appropriate for evaluating the potential deleterious effects of the toxic spill on wildlife of the Dofiana area. Our study on wetland birds high on the aquatic trophic chain sampled within a few months after the toxic spill in the area around Doñana National Park has shown the accumulation of heavy metals. Fourteen months after the mine waste spill, blood samples from white storks and kites collected in the neighborhood of the park and from control birds at reference areas for comparison were examined by fluorescence image analysis after lymphocyte isolation, and by subsequent alkaline single-cell gel (SCG) electrophoresis, known as the Comet assay. Our results indicate that the exposed birds had a significantly increased level of genotoxic damage compared with control animals from noncontaminated locations.

  13. Disintegration of comet nuclei (United States)

    Ksanfomality, Leonid V.


    The breaking up of comets into separate pieces, each with its own tail, was seen many times by astronomers of the past. The phenomenon was in sharp contrast to the idea of the eternal and unchangeable celestial firmament and was commonly believed to be an omen of impending disaster, especially for comets with tails stretching across half the sky. It is only now that we have efficient enough space exploration tools to see comet nuclei and even - in the particular case of small comet Hartley-2 in 2010 - to watch their disintegration stage. There are also other suspected candidates for disintegration in the vast family of comet nuclei and other Solar System bodies.

  14. Physics of comets

    CERN Document Server

    Krishna Swamy, K S


    The study of Comet Halley in 1986 was a tremendous success for cometary science. In March of that year, six spacecrafts passed through Comet Halley as close as 600 km from the nucleus and made the in situ measurements of various kinds. These space missions to Comet Halley and that of the ICE spacecraft to Comet Giacobini-Zinner combined with studies, both ground-based and above the atmosphere, have increased our knowledge of cometary science in a dramatic way.This new edition of Physics of Comets incorporates these new and exciting findings. The emphasis of the book is on the physical processe

  15. Visually observing comets

    CERN Document Server

    Seargent, David A J


    In these days of computers and CCD cameras, visual comet observers can still contribute scientifically useful data with the help of this handy reference for use in the field. Comets are one of the principal areas for productive pro-amateur collaboration in astronomy, but finding comets requires a different approach than the observing of more predictable targets. Principally directed toward amateur astronomers who prefer visual observing or who are interested in discovering a new comet or visually monitoring the behavior of known comets, it includes all the advice needed to thrive as a comet observer. After presenting a brief overview of the nature of comets and how we came to the modern understanding of comets, this book details the various types of observations that can usefully be carried out at the eyepiece of a telescope. Subjects range from how to search for new comets to visually estimating the brightness of comets and the length and orientation of tails, in addition to what to look for in comet heads a...

  16. DNA Comet Assay and Changes in Microflora Load as Screening Methods to Detect Irradiated Food in Egypt

    International Nuclear Information System (INIS)

    Hammad, A.A.; Abo El Nour, S.A.; Ibrahim, H.M.; Osman, M.E.; Abo El- Nasr, A.


    In the present study the microgel electrophoresis of single cells (DNA Comet Assay), and changes in microflora load were applied to detect irradiation treatment of strawberries and fresh-deboned chicken produced in Egypt. Strawberry samples were irradiated at 1.0, 2.0, 3.0 and 4.0 kGy, stored at 4 degree C±1 and analyzed at 0 and 7 days post.-irradiation. Fresh- deboned chicken meat samples were exposed to 2.0, 3.0, 4.0 and 5.0 kGy, stored at 4 degree C±1 and analyzed at 0, 7, 14 and 21 days post-irradiation. After electrophoresis performance, the accridine orange stain slides were seen under fluorescent microscope and the DNA comets were evaluated by photographic and image analysis. Changes in microflora load of irradiated samples were also evaluated. In all irradiated samples, the DNA fragments stretched or migrated out of the cells towards the anode of the agrose gel and appeared as a “comets” with tail. Whereas, DNA comets of all non-irradiated samples were almost intact, round without tail or had very short tail. Values of DNA % in tails and the tail length increased with increasing irradiation dose and storage times. The DNA comet assay could successfully be used to detect radiation treatment of strawberry and deboned-chicken meat samples up to 7 and 21 days post-irradiation, respectively. The absence of gram-negative bacteria and enterobacteriaceae group as well as the very low count of fungi (mostly yeasts) might be considered another evidence of radiation treatment of strawberries and fresh-deboned chicken.

  17. 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.


    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

  18. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas


    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.

  19. Impaired DNA repair as assessed by the ``comet`` assay in patients developing thyroid carcinoma after radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Leprat, F.; Sarasin, A.; Suarez, H.G. [Institut de Recherches sur le Cancer, 94 - Villesuif (France); Leprat, F.; Alapetite, C.; Rosselli, F.; Ridet, A.; Moustacch, E.; Alapetite, C. [Institut Curie, 75 - Paris (France); Schlumberger, M. [Institut Gustave Roussy, 94 - Villejuif (France)


    A defective cellular response to DNA lesions induced be genotoxic agents may be associated to an increased cancer proneness. This has been clearly identified in some rare but extensively studied genetic diseases such as xeroderma pigmentosum (XP), ataxia telangiectasia (AT) and Fanconi anemia (FA). In practical oncology, most patients receive genotoxic therapeutic agents and the presence of so far unidentified sensitive genotypes could account for an increased susceptibility to cancer in a subgroup of exposed patients. The thyroid gland of children is especially sensitive to the carcinogenic effect of ionizing radiation. Evidence for risk is reported even at doses as low as 0.1 Gy, and the excess relative risk to develop a thyroid tumor following a radiation dose of 1 Gy in childhood is of 7.7 [l]. In order to determine if a defect in repair of DNA strand breaks could be involved, as an early step, in the development of secondary thyroid tumors after radiotherapy, we examined, using the alkaline single cell gel electrophoresis assay (SCGE or `comet`), the response to in vitro {gamma}-rays exposure of lymphocytes of a small group of patients who developed thyroid carcinoma after radiotherapy for a primary tumor. Because of its practical advantages, the alkaline comet assay offers the opportunity to question the role of DNA strand beaks rejoining capacity of the individual in the radiation induced carcinogenesis of thyroid tumors. This preliminary study of a small group of patients with therapeutic irradiation at childhood for a primary tumor indicates that, at the time of blood sampling, lymphocytes of some of these patients demonstrated reduced rejoining capacity. These results suggest that the comet assay might help to distinguish a subgroup of individuals at risk for radiation induced genomic instability and encourage further investigation. (authors)

  20. Epigenetics reloaded: the single-cell revolution. (United States)

    Bheda, Poonam; Schneider, Robert


    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.

  1. Single cell enzyme diagnosis on the chip

    DEFF Research Database (Denmark)

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


    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 detec...

  2. Radio response of human lymphocytes pretreated with boron and gadoliniums assessed by the, comet assay

    International Nuclear Information System (INIS)

    Kim, J. K.; Park, T. W.; Cebulska-Wasiewska, A.; Nili, M.


    Boron and gadolinium are among the nuclides that hold a unique property of being a neutron capture therapy agent. Neutron beams have often a considerable portion of gamma rays with fast neutrons. Gamma rays, as beam contaminants, can cause considerable damage to normal tissues even if such tissues do contain high boron concentrations. Materials and Methods: The modification of radio response in human lymphocytes pretreated with boron or gadolinium compound was studied by assessing the DNA damage using single cell gel electrophoresis, the comet assay. The lymphocytes from the human peripheral blood were irradiated with 0, 1, 2 and 4 Gy of gamma rays from a 60 Co isotopic source with or without pretreatment of boron or gadolinium compound for 10 minutes at 4 d egree C . Post-irradiation procedures included slide preparation, cell-lysing, unwinding and electrophoresis, neutralization, staining, and analytic steps, gel electrophoresis. Results: The results indicate that pretreatment with boron compound (50 n M or 250 n M of 10 B) is effective in reducing the radiosensitivity of the lymphocyte DNA. Conversely, pretreatment with gadolinium compound (50 n M) led to a dose-dependent increase in the radiosensitivity, most prominently with a dose of 4 Gy (P<0.001). Furthermore, when the lymphocytes were pretreated with a Combined mixture (1:1) of boron (250 n M) and gadolinium (50 n M) compounds, the reduced radiosensitivity was also observed.

  3. Ammonia abundances in comets (United States)

    Wyckoff, S.; Tegler, S.; Engel, L.

    The emission band strengths of the NH2 bands of Comets Halley, Hartley-Good, Thiele, and Borrelly were measured to determine the NH2 column densities for the comets. Production rates obtained using the Haser and vectorial models are in agreement within the observational errors, suggesting that a simple two-step decay model may be used to approximate the NH2 distribution in a comet's coma. Ammonia-to-water abundance ratios from 0.01 to 0.4 percent were found for the four comets. The ratio in Comet Halley is found to be Q(NH3)/Q(H2O) = 0.002 + or - 0.001. No significant difference in the ammonia abundance was found before or after perihelion in Comet Halley.

  4. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma


    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  5. Special Report: Chemistry of Comets. (United States)

    A'Hearn, Michael F.


    Discusses the chemistry of comets. How comets provide clues to the birth of the solar system, photolytic reactions on comets involving water, chemical modeling, nuclear chemistry, and research findings are among the areas considered. (JN)

  6. Technologies for Single-Cell Isolation. (United States)

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


    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.

  7. Detection of the irradiation treatment of foods using micro gel electrophoresis of DNA

    International Nuclear Information System (INIS)

    Delincee, H.


    Ionizing radiation has profound effects on nucleic acids, reflected by e.g. base damage and strandbreaks. These effects of radiation contribute to the aim of food irradiation: undesirable microorganisms are inactivated, insect infestation eliminated, sprouting inhibited or ripening delayed. It is, therefore, conceivable that methods of detection of a radiation treatment could be based on changes in DNA, either in microbes or insects or in the food itself. A promising method of detecting DNA fragments in irradiated food is the microelectrophoresis of single cells. Advantages of the method are its simplicity and its speed, the electrophoretic separation only requiring 2.5-5 minutes. The principle is based on migration of DNA in an agarose gel exposed to an electric field. Single cells or nuclei are embedded in the agarose and after lysis intact DNA will practically not move upon electrophoresis, due to the gel strains, whereas if DNA fragmentation has occurred, the DNA is able to migrate and ''comets'' following the cells or nuclei become visible after staining. This paper describes our experience obtained by analyzing chicken, beef and pork meat, shrimps and mushrooms. The method therefore has its limitations, although good results both from frozen and fresh meats, fresh fish, onions and potatoes have been reported. Also strawberries have been identified using this technique. Further experience may strengthen the evidence that this simple and cheap technique may have its place among the battery of tests to detect the radiation treatment of foods. (orig./vhe)

  8. Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana Ecological Disaster. (United States)

    Pastor, N; López-Lázaro, M; Tella, J L; Baos, R; Hiraldo, F; Cortés, F


    Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Doñana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.

  9. Genotoxicity determinations of coriander drop and extract of Coriander Sativum cultured fibroblast of rat embryo by comet assay

    International Nuclear Information System (INIS)

    Heibatullah, K.; Marzieh, P.; Arefeh, I.; Ebrahim, M.


    The single cell gel electrophoresis (SCGE) or comet assay is a quick, simple and sensitive technique for measuring DNA damage in cell nucleus. It is well known that medicinal herbs play an important role in the life of human beings, thus it is essential to determine their safety as public health is concerned. In this study the genotoxicity of Coriander drop, herbal pharmaceutical product, and the extract of Coriander sativum were examined in cultured fibroblast of rat embryo using comet assay. The thirteen to fifteen days old rat embryos were lysed with tripsin and after certain steps it was centrifuged and then cultured. After three to five passages, different concentrations of each product were applied to the fibroblasts. Lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. In the test groups the results indicated that coriander drop at different doses showed some fragmentation of DNA but this damage as a result was deemed to be not significant. However, in the case of Coriander sativum extract the results showed no mutagenic effects in comparison with the positive control group (p<0.05). In conclusion, these herbal products did not show any magnetic effect according to our test, but further genotoxicity assays are recommended. (author)

  10. Changing Speed of Comets (United States)

    Follows, Mike


    It is shown that highly elliptical orbits, such as those of comets, can be explained well in terms of energy rather than forces. The principle of conservation of energy allows a comet's velocity to be calculated at aphelion and perihelion. An example asks students to calculate whether they can run fast enough to escape from a small asteroid.…

  11. Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting. (United States)

    Duncombe, Todd A; Kang, Chi-Chih; Maity, Santanu; Ward, Toby M; Pegram, Mark D; Murthy, Niren; Herr, Amy E


    Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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


    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. Subcellular western blotting of single cells. (United States)

    Yamauchi, Kevin A; Herr, Amy E


    Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

  14. Realm of the comets

    International Nuclear Information System (INIS)

    Weissman, P.R.


    Studies of Jovian perturbations of the orbits of long-period comets led to the concept of the Oort cloud of 180 billion comets at 50,000-150,000 AU from the sun. Several comets are induced to move toward the sun every million years by the passage of a star at a distance of a few light years. The location of the cloud has since been revised to 20,000-100,000 AU, and comets are now accepted as remnant material fron the proto-solar system epoch. The galactic disk and random, close-passing stars may also cause rare, large perturbations in the orbits of the cloud comets, sending large numbers of comets through the inner solar system. The resulting cometary storm is a candidate cause for the wholesale extinction of dinosaurs in the Cretaceous-Terniary transition due to large number of planetesimals, or one large comet, striking the earth, in a short period of time. The IRAS instruments have detected similar clouds of material around other stars

  15. DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD). (United States)

    Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária


    3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Comets in UV (United States)

    Shustov, B.; Sachkov, M.; Gómez de Castro, A. I.; Vallejo, J. C.; Kanev, E.; Dorofeeva, V.


    Comets are important "eyewitnesses" of Solar System formation and evolution. Important tests to determine the chemical composition and to study the physical processes in cometary nuclei and coma need data in the UV range of the electromagnetic spectrum. Comprehensive and complete studies require additional ground-based observations and in situ experiments. We briefly review observations of comets in the ultraviolet (UV) and discuss the prospects of UV observations of comets and exocomets with space-borne instruments. A special reference is made to the World Space Observatory-Ultraviolet (WSO-UV) project.

  17. Comets: looking ahead. (United States)

    A'Hearn, Michael F


    We outline the key questions about comets that must be answered in order to understand cometary formation in the context of the protoplanetary disc and the role of comets in the formation and evolution of the solar system. We then discuss the new understanding of comets from Rosetta and from other recent advances, including work presented by others at the discussion meeting. Finally, we suggest some key directions for future projects to better address the above questions.This article is part of the themed issue 'Cometary science after Rosetta'. © 2017 The Author(s).

  18. Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response. (United States)

    Kang, Chi-Chih; Lin, Jung-Ming G; Xu, Zhuchen; Kumar, Sanjay; Herr, Amy E


    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

  19. DIRBE Comet Trails (United States)

    Arendt, Richard G.


    Re-examination of the COBE DIRBE data reveals the thermal emission of several comet dust trails.The dust trails of 1P/Halley, 169P/NEAT, and 3200 Phaethon have not been previously reported.The known trails of 2P/Encke, and 73P/Schwassmann-Wachmann 3 are also seen. The dust trails have 12 and 25 microns surface brightnesses of comet, but others are best seen at high ecliptic latitudes as the Earth passes over or under the dust trail. All these comets have known associations with meteor showers. This re-examination also reveals one additional comet and 13 additional asteroids that had not previously been recognized in the DIRBE data.

  20. William Herschel and Comets (United States)

    Sullivan, Woodruff


    I examine the observational and theoretical researches of William Herschel on 21 comets that he observed over the period 1781 to 1812. Herschel's focus, unlike most contemporaries, was on their physical structure, not their orbits. He forged a strong connection between comets and his nebulae with a scheme of cometary "maturation" (1812) involved a comet traveling from star to star after its central "planetary body'; was born from gravitational collapse of a nebula. During close passages of a star, the comet brightened and lost mass from its atmosphere; at other times, when between stars, it encountered nebulae and was rejuvenated by picking up more mass. Laplace soon adopted these ideas to improve his nebula hypothesis for solar system formation.

  1. Optimization of genetic analysis for single cell

    Directory of Open Access Journals (Sweden)

    hussein mouawia


    Full Text Available The molecular genetic analysis of microdissected cells by laser, a method for selecting a starting material of pure DNA or RNA uncontaminated. Our study focuses on technical pre-PCR (polymerase chain reaction for the amplification of DNA from a single cell (leukocyte isolated from human blood after laser microdissection and aims to optimize the yield of DNA extracted of this cell to be amplified without errors and provide reliable genetic analyzes. This study has allowed us to reduce the duration of cell lysis in order to perform the step of expanding genomic PEP (primer extension preamplification directly after lysis the same day and the quality of genomic amplification and eliminate purification step of the product PEP, step with a risk of contamination and risk of loss of genetic material related to manipulation. This approach has shown that the combination of at least 3 STR (short tandem repeat markers for genetic analysis of single cell improves the efficiency and accuracy of PCR and minimizes the loss of allele (allele drop out; ADO. This protocol can be applied to large scale and an effective means suitable for genetic testing for molecular diagnostic from isolated single cell (cancerous - fetal.

  2. Geometry-induced injection dispersion in single-cell protein electrophoresis. (United States)

    Pan, Qiong; Herr, Amy E


    Arrays of microwells are widely used to isolate individual cells, facilitate high throughput cytometry assays, and ensure compatibility of those assays with whole-cell imaging. Microwell geometries have recently been utilized for handling and preparation of single-cell lysate, prior to single-cell protein electrophoresis. It is in the context of single-cell electrophoresis that we investigate the interplay of microwell geometry (circular, rectangular, triangular) and transport (diffusion, electromigration) on the subsequent performance of single-cell polyacrylamide gel electrophoresis (PAGE) for protein targets. We define and measure injector-induced dispersion during PAGE, and develop a numerical model of band broadening sources, experimentally validate the numerical model, and then identify operating conditions (characterized through the Peclet number, Pe) that lead to microwell-geometry induced losses in separation performance. With analysis of mammalian cells as a case study, we sought to understand at what Pe is the PAGE separation performance adversely sensitized to the microwell geometry. In developing design rules, we find that for the microwell geometries that are the most suitable for isolation of mammalian cells and moderate mass protein targets, the Pe is usually small enough (Pe geometry on protein PAGE of single-cell lysate. In extreme cases where the largest mammalian cells are analyzed (Pe > ∼20), consideration of Pe suggests using a rectangular - and not the widely used circular - microwell geometry to maximize protein PAGE separation performance. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Comets in Australian Aboriginal Astronomy (United States)

    Hamacher, Duane W.; Norris, Ray P.


    We present 25 accounts of comets from 40 Australian Aboriginal communities, citing both supernatural perceptions of comets and historical accounts of historically bright comets. Historical and ethnographic descriptions include the Great Comets of 1843, 1861, 1901, 1910, and 1927. We describe the perceptions of comets in Aboriginal societies and show that they are typically associated with fear, death, omens, malevolent spirits, and evil magic, consistent with many cultures around the world. We also provide a list of words for comets in 16 different Aboriginal languages.

  4. The use of comet assay in plant toxicology: recent advances

    Directory of Open Access Journals (Sweden)

    Conceição LV Santos


    Full Text Available The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g. Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage.

  5. The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C.

    Directory of Open Access Journals (Sweden)

    J Strzezek


    Full Text Available The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis, to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G were diluted with a standard semen extender, Kortowo-3 (K-3, which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh or ostrich egg yolk (LPFo. Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional

  6. Thermoresponsive micropatterned substrates for single cell studies.

    Directory of Open Access Journals (Sweden)

    Kalpana Mandal

    Full Text Available We describe the design of micropatterned surfaces for single cell studies, based on thermoresponsive polymer brushes. We show that brushes made of poly(N-isopropylacrylamide grafted at high surface density display excellent protein and cell anti-adhesive properties. Such brushes are readily patterned at the micron scale via deep UV photolithography. A proper choice of the adhesive pattern shapes, combined with the temperature-dependent swelling properties of PNIPAM, allow us to use the polymer brush as a microactuator which induces cell detachment when the temperature is reduced below [Formula: see text]C.

  7. Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks; Evaluation des Comet Assays bei neutralem pH zur Detektion von α-Partikel induzierten DNA-Doppelstrangbruechen

    Energy Technology Data Exchange (ETDEWEB)

    Hofbauer, Daniela


    Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

  8. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg


    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  9. Protective effects of aqueous and ethanolic extracts of Portulaca oleracea L. aerial parts on H2O2-induced DNA damage in lymphocytes by comet assay. (United States)

    Behravan, Javad; Mosafa, Fatemeh; Soudmand, Negar; Taghiabadi, Elahe; Razavi, Bibi Marjan; Karimi, Gholamreza


    The comet assay is a standard method for measuring DNA damage. In this study, the protective effects of ethanolic and aqueous extracts of Portulaca oleracea L. (P. oleracea) on human lymphocyte DNA lesions were evaluated with the comet assay. Lymphocytes were isolated from blood samples taken from healthy volunteers. Human lymphocytes were incubated in H(2)O(2) (50,100, and 200 μM), aqueous extract (0.05, 0.1, 0.5, 1, and 2.5mg/ml), and ethanolic extracts (0.05, 0.1, 0.5, 1, and 2.5mg/ml) of P. oleraceae aerial parts alone with a combination of H(2)O(2) (100 μM) with either 1 or 2.5mg/ml of both extracts at 4°C for 30 minutes. The extent of DNA migration was measured using the alkaline single cell gel electrophoresis approach assay, and DNA damage was expressed as percentage tail DNA. We found that the aqueous extract of P. oleracea significantly inhibited DNA damage, while there was no effect of the ethanolic extract. These data suggest that the aqueous extract of P. oleracea can prevent oxidative DNA damage to human lymphocytes, which is likely due to antioxidant constituents in the extract. Copyright © 2011. Published by Elsevier B.V.

  10. Jupiter Laser Facility - COMET Laser (United States)

    Federal Laboratory Consortium — COMET has 4 beam configurations with uncompressed pulse lengths from 500 ps to 6 ns, compressed pulses to 0.5 ps, and beam energies up to 20 J. COMET can fire every...

  11. Evaluation of the neutral comet assay for detection of alpha-particle induced DNA-double-strand-breaks

    International Nuclear Information System (INIS)

    Hofbauer, Daniela


    Aim of this study was to differentiate DNA-double-strand-breaks from DNA-single-strand-breaks on a single cell level, using the comet assay after α- and γ-irradiation. Americium-241 was used as a alpha-irradiation-source, Caesium-137 was used for γ-irradiation. Because of technical problems with both the neutral and alkaline comet assay after irradiation of gastric cancer cells and human lymphocytes, no definite differentiation of DNA-damage was possible.

  12. Discovering the Nature of Comets. (United States)

    Whipple, Fred L.


    "The Mystery of Comets" by Dr. Fred Whipple provides an introduction to the modern picture of comets and his personal reminiscences of how his model of comets came to be. An adaptation of several sections of the book is presented. (JN)

  13. Term Projects on Interstellar Comets (United States)

    Mack, John E.


    Presents two calculations of the probability of detection of an interstellar comet, under the hypothesis that such comets would escape from comet clouds similar to that believed to surround the sun. Proposes three problems, each of which would be a reasonable term project for a motivated undergraduate. (Author/MLH)

  14. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Fung, N.


    Since the first demonstration of the laser in the 1960`s, lasers have found numerous applications in analytical chemistry. In this work, two different applications are described, namely, DNA sequencing with capillary gel electrophoresis and single cell analysis with mass spectrometry. Two projects are described in which high-speed DNA separations with capillary gel electrophoresis were demonstrated. In the third project, flow cytometry and mass spectrometry were coupled via a laser vaporization/ionization interface and individual mammalian cells were analyzed. First, DNA Sanger fragments were separated by capillary gel electrophoresis. A separation speed of 20 basepairs per minute was demonstrated with a mixed poly(ethylene oxide) (PEO) sieving solution. In addition, a new capillary wall treatment protocol was developed in which bare (or uncoated) capillaries can be used in DNA sequencing. Second, a temperature programming scheme was used to separate DNA Sanger fragments. Third, flow cytometry and mass spectrometry were coupled with a laser vaporization/ionization interface.

  15. Death of a comet

    CERN Multimedia

    Hawkes, N


    The comet Linear dissolved as it made its closest approach to the sun on July 25th. The first stages of its breakup had been witnessed by the Hubble telescope when it threw off a piece of its crust (3 paragraphs).

  16. Comet radar explorer (United States)

    Farnham, Tony; Asphaug, Erik; Barucci, Antonella; Belton, Mike; Bockelee-Morvan, Dominique; Brownlee, Donald; Capria, Maria Teresa; Carter, Lynn; Chesley, Steve; Farnham, Tony; Gaskell, Robert; Gim, Young; Heggy, Essam; Herique, Alain; Klaasen, Ken; Kofman, Wlodek; Kreslavsky, Misha; Lisse, Casey; Orosei, Roberto; Plaut, Jeff; Scheeres, Dan

    The Comet Radar Explorer (CORE) is designed to perform a comprehensive and detailed exploration of the interior, surface, and inner coma structures of a scientifically impor-tant Jupiter family comet. These structures will be used to investigate the origins of cometary nuclei, their physical and geological evolution, and the mechanisms driving their spectacular activity. CORE is a high heritage spacecraft, injected by solar electric propulsion into orbit around a comet. It is capable of coherent deep radar imaging at decameter wavelengths, high resolution stereo color imaging, and near-IR imaging spectroscopy. Its primary objective is to obtain a high-resolution map of the interior structure of a comet nucleus at a resolution of ¿100 elements across the diameter. This structure shall be related to the surface geology and morphology, and to the structural details of the coma proximal to the nucleus. This is an ideal complement to the science from recent comet missions, providing insight into how comets work. Knowing the structure of the interior of a comet-what's inside-and how cometary activity works, is required before we can understand the requirements for a cryogenic sample return mission. But more than that, CORE is fundamental to understanding the origin of comets and their evolution in time. The mission is made feasible at low cost by the use of now-standard MARSIS-SHARAD reflec-tion radar imaging hardware and data processing, together with proven flight heritage of solar electric propulsion. Radar flight heritage has been demonstrated by the MARSIS radar on Mars Express (Picardi et al., Science 2005; Plaut et al., Science 2007), the SHARAD radar onboard the Mars Reconnaissance Orbiter (Seu et al., JGR 2007), and the LRS radar onboard Kaguya (Ono et al, EPS 2007). These instruments have discovered detailed subsurface structure to depths of several kilometers in a variety of terrains on Mars and the Moon. A reflection radar deployed in orbit about a comet

  17. Comets in Indian Scriptures (United States)

    Das Gupta, P.


    The Indo-Aryans of ancient India observed stars and constellations for ascertaining auspicious times in order to conduct sacrificial rites ordained by the Vedas. Naturally, they would have sighted comets and referred to them in the Vedic texts. In Rigveda (circa 1700-1500 BC) and Atharvaveda (circa 1150 BC), there are references to dhumaketus and ketus, which stand for comets in Sanskrit. Rigveda speaks of a fig tree whose aerial roots spread out in the sky (Parpola 2010). Had this imagery been inspired by the resemblance of a comet's tail with long and linear roots of a banyan tree (ficus benghalensis)? Varahamihira (AD 550) and Ballal Sena (circa AD 1100-1200) described a large number of comets recorded by ancient seers, such as Parashara, Vriddha Garga, Narada, and Garga, to name a few. In this article, we propose that an episode in Mahabharata in which a radiant king, Nahusha, who rules the heavens and later turns into a serpent after he kicked the seer Agastya (also the star Canopus), is a mythological retelling of a cometary event.

  18. Halley's Comet: A Bibliography. (United States)

    Freitag, Ruth S., Comp.

    Included in this bibliography are over 3,200 references to publications on Halley's Comet, its history, orbital motion, and physical characteristics, meteor streams associated with it, preparations for space missions to study it in 1986, and popular reaction to its appearances. Also cited are a few papers that, although they devote little…

  19. Splitting of Comets

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 1. Splitting of Comets. Utpal Mukhopadhyay. General Article Volume 7 Issue 1 January 2002 pp 11-22. Fulltext. Click here to view fulltext PDF. Permanent link: Keywords. Cometary ...

  20. Comet 2001 Q2

    Czech Academy of Sciences Publication Activity Database

    Pravec, Petr; Kušnirák, Peter; Bouma, R. J.; Raymundo, P. M.

    č. 7687 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  1. Asteroids, meteorites, and comets

    CERN Document Server

    Elkins-Tanton, Linda T


    Asteroids, Comets, and Meteorites provides students, researchers, and general readers with the most up-to-date information on this fascinating field. From the days of the dinosaurs to our modern environment, this book explores all aspects of these cosmic invaders.

  2. DRBE comet trails

    Energy Technology Data Exchange (ETDEWEB)

    Arendt, Richard G., E-mail: [CREST/UMBC, Code 665, NASA/GSFC, Greenbelt, MD 20771 (United States)


    Re-examination of the Cosmic Background Explorer Diffuse Infrared Background Experiment (DIRBE) data reveals the thermal emission of several comet dust trails. The dust trails of 1P/Halley, 169P/NEAT, and 3200 Phaethon have not been previously reported. The known trails of 2P/Encke and 73P/Schwassmann–Wachmann 3 are also seen. The dust trails have 12 and 25 μm surface brightnesses of <0.1 and <0.15 MJy sr{sup −1}, respectively, which is <1% of the zodiacal light intensity. The trails are very difficult to see in any single daily image of the sky, but are evident as rapidly moving linear features in movies of the DIRBE data. Some trails are clearest when crossing through the orbital plane of the parent comet, but others are best seen at high ecliptic latitudes as the Earth passes over or under the dust trail. All these comets have known associations with meteor showers. This re-examination also reveals 1 additional comet and 13 additional asteroids that had not previously been recognized in the DIRBE data.

  3. A Search of Reactivated Comets (United States)

    Ye, Quan-Zhi


    Dormant or near-dormant short-period comets can unexpectedly regain the ability to eject dust. In many known cases, the resurrection is short-lived and lasts less than one orbit. However, it is possible that some resurrected comets can remain active in later perihelion passages. We search the archival images of various facilities to look for these “reactivated” comets. We identify two candidates, 297P/Beshore and 332P/Ikeya-Murakami, both of which were found to be inactive or weakly active in the previous orbit before their discovery. We derive a reactivation rate of ˜ 0.007 {{comet}}-1 {{orbit}}-1, which implies that typical short-period comets only become temporarily dormant a few times or less. Smaller comets are prone to rotational instability and may undergo temporary dormancy more frequently. Next generation high-cadence surveys may find more reactivation events of these comets.

  4. Cometography a catalog of comets

    CERN Document Server

    Kronk, Gary W; Seargent, David A J


    Cometography is a multi-volume catalog of every comet observed from ancient times up to the 1990s, when the internet took off as a medium of scientific record. It uses the most reliable orbits known to determine the distances from the Earth and Sun at the time of discovery and last observation, as well as the largest and smallest angular distance to the Sun, most northerly and southerly declination, closest distance to the Earth, and other details, to enable the reader to understand each comet's physical appearance. Volume 6, the final volume in the catalog, covers the observations and pertinent calculations for every comet seen between 1983 and 1993. The comets are listed in chronological order, with complete references to publications relating to each comet and physical descriptions of each comet's development throughout its apparition. Cometography is the definitive reference on comets through the ages, for astronomers and historians of science.

  5. Assessment of status of three water bodies in Serbia based on tissue metal and metalloid concentration (ICP-OES) and genotoxicity (comet assay). (United States)

    Sunjog, Karolina; Kolarević, Stoimir; Kračun-Kolarević, Margareta; Višnjić-Jeftić, Željka; Skorić, Stefan; Gačić, Zoran; Lenhardt, Mirjana; Vasić, Nebojša; Vuković-Gačić, Branka


    Metals and metalloids are natural components of the biosphere, which are not produced per se by human beings, but whose form and distribution can be affected by human activities. Like all substances, they are a contaminant if present in excess compared to background levels and/or in a form that would not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub, Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer Software (Perceptive Instruments, UK). The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by: (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of European chub: liver, gills, muscle and gonads (ii) comparing these findings with genotoxicity of water expressed through DNA damage of fish tissues. A clear link between the level of metals in water, sediment and tissues and between metal and genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the organic compounds), contribute to DNA damage. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. 67P, Singing Comet (United States)

    Smirnova, Ekaterina


    I would like to propose to present a short science-art-music collaboration film called "67P, Singing Comet" (5:27 min). If time of the session will allow, prior to the film I would like to make a slide show introduction to this project, highlighting the inspiration - the mission Rosetta by the European Space Agency (ESA) - and the artistic collaboration that took place in creating this piece. Inspired by the ESA Rosetta mission to the comet 67P, Ekaterina Smirnova (artist and project director, New York), Lee Mottram (clarinetist, Wales), Takuto Fukuda (composer, Japan) and Brian Hekker (video editor, New York) collaborated to create a unique atmospheric piece. Water and the origins of life throughout the Universe (specifically the Earth) is an element of the mission and the focus of Ekaterina's artistic vision. Ekaterina literally and figuratively paints a sensory assemblage using a combination of synthetic and natural elements to shape this artistic creation. To paint her watercolor works she is using a replica of the water found on the comet and implementing her own heartbeat into the music to create a recognizable inward sound of life. The Electro-Acoustic composition by Takuto Fukuda features an electronically manipulated performance by clarinetist Lee Mottram. The piece ceremoniously begins with reverberant bursts of low-register atonal bells transporting the listener to their ethereal inner origins of body and mind. The imagination takes the experience to an unknown destination as it gains speed gliding through the visual and audible textures of space and time. The comet's water similarly reacts with an ebb and flow thawing ice to potentially give life a chance as it is thrust along an orbit around the Sun. Near then far from the heat the comet forms frozen particles from vapors as it reaches it's furthest stretches creating an aerodynamic tail of icicles that slowly dissipate in a cycle that repeats itself until the comet's ultimate collision with an

  7. Irradiation detection of food by DNA Comet Assay

    International Nuclear Information System (INIS)

    Khan, A.A.; Delincee, H.


    Microgel electrophoresis of single cells or nuclei (DNA Comet Assay) has been investigated to detect irradiation treatment of more than 50 food commodities e.g. meats, seafood, cereals, pulses, nuts, fruits and vegetables, and spices. The foodstuffs have been exposed to radiation doses covering the range of potential commercial irradiation for inactivation of pathogenic and spoilage micro-organisms, for insect disinfestation and for shelf-life extension. The Comet Assay is based on detection of DNA fragments presumptive to irradiation. For most of the food items investigated, the assay can be applied successfully for irradiation detection by working out different conditions of the assay. However, with some of the foods difficulties arose due to - lack of discrimination between the irradiated and unirradiated food samples due to the presence of the same kinds of comets in both cases and the total absence of the typical intact cells in unirradiated samples. - Sufficient DNA material was not available from some of the foods. - Insufficient lysis of the cell walls in case of some plant foods. In conclusion, the DNA Comet Assay can help to detect the irradiation treatment of several varieties of foods using low-cost equipment in a short time of analysis. (orig.)

  8. Gravisensing in single-celled systems (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

  9. Fluorescent in situ hybridization on comets: FISH comet. (United States)

    Shaposhnikov, Sergey; El Yamani, Naouale; Collins, Andrew R


    The DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. The comet assay, a sensitive method for monitoring DNA damage and repair, involves electrophoresis of nucleoids comprising supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. We use fluorescent in situ hybridization (FISH) to investigate the structure of the chromatin within comet preparations and to study specific DNA sequences within comets. In this chapter we describe our FISH comets protocols, deal with some technical questions and outline the theory. FISH with comets should be useful to researchers interested in the structural organization of DNA and chromatin, the localization of DNA damage, and the kinetics of repair of damage.

  10. CINE: Comet INfrared Excitation (United States)

    de Val-Borro, Miguel; Cordiner, Martin A.; Milam, Stefanie N.; Charnley, Steven B.


    CINE calculates infrared pumping efficiencies that can be applied to the most common molecules found in cometary comae such as water, hydrogen cyanide or methanol. One of the main mechanisms for molecular excitation in comets is the fluorescence by the solar radiation followed by radiative decay to the ground vibrational state. This command-line tool calculates the effective pumping rates for rotational levels in the ground vibrational state scaled by the heliocentric distance of the comet. Fluorescence coefficients are useful for modeling rotational emission lines observed in cometary spectra at sub-millimeter wavelengths. Combined with computational methods to solve the radiative transfer equations based, e.g., on the Monte Carlo algorithm, this model can retrieve production rates and rotational temperatures from the observed emission spectrum.

  11. Diatoms in comets (United States)

    Hoover, R.; Hoyle, F.; Wallis, M. K.; Wickramasinghe, N. C.


    The fossil record of the microscopic algae classified as diatoms suggests they were injected to earth at the Cretaceous boundary. Not only could diatoms remain viable in the cometary environment, but also many species might replicate in illuminated surface layers or early interior layers of cometary ice. Presumably they reached the solar system on an interstellar comet as an already-evolved assemblage of organisms. Diatoms might cause color changes to comet nuclei while their outgassing decays and revives around highly elliptical orbits. Just as for interstellar absorption, high-resolution IR observations are capable of distinguishing whether the 10-micron feature arises from siliceous diatom material or mineral silicates. The 10-30-micron band and the UV 220-nm region can also provide evidence of biological material.

  12. The potential of single-cell profiling in plants. (United States)

    Efroni, Idan; Birnbaum, Kenneth D


    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

  13. Micro-PIXE for single cell analysis

    International Nuclear Information System (INIS)

    Ortega, Richard


    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)

  14. Comet Radar Explorer (United States)

    Asphaug, Erik; CORE Science Team


    Comet Radar Explorer (CORE) is a low cost mission that uses sounding radar to image the 3D internal structure of the nucleus of Jupiter-family comet (JFC) Tempel 2. Believed to originate in the Kuiper Belt, JFCs are among the most primitive bodies in the inner solar system. CORE operates a 5 and 15 MHz Radar Reflection Imager from close orbit about the nucleus of Tempel 2, obtaining a dense network of echoes that are used to map its interior dielectric contrasts to high resolution (ង m) and resolve the dielectric constants to  m throughout the 16x8x9 km nucleus. The resulting clear images of internal structure and composition reveal how the nucleus was formed and how it has evolved. Radiometric tracking of the spacecraft orbit results in an interior mass distribution that constrains the radar-based models of interior composition. High-resolution visible and infrared color images provide the surface and exterior boundary conditions for interior models and hypotheses. They present the geology and morphology of the nucleus surface at meter-scales, and also the time-evolving activity, structure and composition of the inner coma. By making deep connections from interior to exterior, the data CORE provides will answer fundamental questions about the earliest stages of planetesimal evolution and planet formation, and lay the foundation for a comet nucleus sample return mission. CORE is led by Prof. Erik Asphaug of the University of California, Santa Cruz and is managed by JPL. It benefits from key scientific and payload contributions by ASI and CNES. The international science team has been assembled on the basis of their key involvement in past and ongoing missions to comets, and in Mars radar missions, and for their expertise in radar data analysis.

  15. Comets and their origin the tools to decipher a comet

    CERN Document Server

    Meierhenrich, Uwe


    Divided into two parts, the first four chapters of Comets and their Origin refer to comets and their formation in general, describing cometary missions, comet remote observations, astrochemistry, artificial comets, and the chirality phenomenon.The second part covers the cometary Rosetta mission, its launch, journey, scientific objectives, and instrumentations, as well as the landing scenario on a cometary nucleus. Along the way, the author presents general questions concerning the origin of terrestrial water and the molecular beginnings of lifeon Earth, as well as how the instruments used on

  16. Silicon Dioxide Thin Film Mediated Single Cell Nucleic Acid Isolation (United States)

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


    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

  17. The comet assay – from toy to tool

    Directory of Open Access Journals (Sweden)

    Guenter Speit


    Full Text Available The comet assay is nowadays the most common method for measuring DNA damage and repair in single cells. It is based on the microelectrophoretic study published by Ostling and Johanson (1984 and was developed by Singh and coworkers (1988 to a versatile technique for quantitation of low levels of DNA damage in individual cells. This alkaline version still is the basis for the triumphant success of the comet assay in basic research into mechanisms of DNA damage and DNA repair, genotoxicity testing, ecotoxicology and human biomonitoring. Important technical improvements (e.g., the use of precoated slides, introduction of image analysis, high throughput methods, automated scoring systems made the assay more robust and more efficient. Modifications of the standard protocol provide more specific information on the type and biological significance of the damage studied. The introduction of lesion-specific endonucleases allowed the characterization of oxidative base damage, alkylation damage and UV-induced pyrimidine dimers. The combination with fluorescence in situ hybridization (FISH made it possible to identify DNA of particular chromosome regions and measure effects of damage and repair in particular genes. The comet assay is being increasingly used in genotoxicity testing. In particular, the in vivo comet assay has become a component of some genotoxicity test strategies and generally accepted test protocols have evolved over the years. A large international collaborative trial sponsored by the Japanese Center for the Validation of Alternative Methods (JaCVAM was recently completed and an OECD test guideline was approved. The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. However, there are still problems in comparing results from different laboratories and there is need for reducing inter-laboratory variation and identification of standard


    African Journals Online (AJOL)


    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae ... animal feed but little or no information has been documented as per its explication for the production of single cell .... use of yeasts produced from vatious carbohydrate sources, molasses, sulphite liquors and vegetable.

  19. Design and Analysis of Single-Cell Sequencing Experiments

    NARCIS (Netherlands)

    Grün, Dominic; van Oudenaarden, Alexander


    Recent advances in single-cell sequencing hold great potential for exploring biological systems with unprecedented resolution. Sequencing the genome of individual cells can reveal somatic mutations and allows the investigation of clonal dynamics. Single-cell transcriptome sequencing can elucidate

  20. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

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

    Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can...... 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...

  1. Clinical Outcome Prediction Using Single-Cell Data. (United States)

    Pouyan, Maziyar Baran; Jindal, Vasu; Nourani, Mehrdad


    Single-cell technologies like flow cytometry (FCM) provide valuable biological data for knowledge discovery in complex cellular systems like tissues and organs. FCM data contains multi-dimensional information about the cellular heterogeneity of intricate cellular systems. It is possible to correlate single-cell markers with phenotypic properties of those systems. Cell population identification and clinical outcome prediction from single-cell measurements are challenging problems in the field of single cell analysis. In this paper, we propose a hybrid learning approach to predict clinical outcome using samples' single-cell FCM data. The proposed method is efficient in both i) identification of cellular clusters in each sample's FCM data and ii) predict clinical outcome (healthy versus unhealthy) for each subject. Our method is robust and the experimental results indicate promising performance.

  2. Technical manual for COMET

    International Nuclear Information System (INIS)

    Song, Jin Ho; Kwon, Young Min; Kim, Taek Mo; Lee, Sang Jong; Jeong, Hae Yong


    The purpose of this report is to provide a description for a COMET computer code which is to be used in the analysis of mass and energy releases during post-blowdown phase of LOCA. The mass and energy data re to be used as input data for the containment functional design. This report contains a brief description of analytical models and guidelines for the usage of the computer code. This computer code is to be used for both cold leg and hot leg break analyses. A verification analyses are performed for Ulchin 3 and 4 cold and hot leg break. 11 figs (Author)

  3. Physics of comets

    International Nuclear Information System (INIS)

    Swamy, K.S.K.


    This book deals systematically with the physics of comets in the light of our present knowledge about various aspects of cometary phenomena and the problems that require further attention. The basic aspects, methods and models that have been used extensively at the present time for the interpretation of cometary observations are fully discussed. Contents: General Introduction; Orbital Dynamics; Atomic Physics; Spectra; Spectra of Coma; Gas Production Rates; Dust Tail; Light Scattering Theory; Nature of the Dust Particle; Ion Tails; Nucleus; Origins; Problems and Prospects

  4. Inside look at Halley's comet

    International Nuclear Information System (INIS)

    Beatty, J.K.


    The 1985-1986 emergence of Halley's comet, the first since the advent of the space age, was explored by a variety of spacecraft. The Vega 1, launched by the USSR together with the Eastern-block alliance, passed 5523 miles from the comet's nucleus at 7:20:06 Universal time. It indicated that the comet was about 300 miles closer to the sun than had been predicted. The Japanese spacecraft, Suisei, was created to map the distribution of neutral hydrogen atoms outside Halley's visible coma. Its pictures indicated that the comet's output of water varied between 25 and 60 tons per second. Five days after the Vega 2's passage through the comet, the Giotto (sponsored by the European Space Agency) probe appeared. Giotto's close approach took place 3.1 minutes after midnight UT on March 14th; the craft had passed 376 miles from its target. Giotto's data indicated that the nucleus was bigger than expected, and that the comet was composed primarily of water, CO2 and N2. The Vegas and Giotto found that as the solar wind approaches Halley, it slows gradually and the solar magnetic lines embedded in the wind begin to pile up. Pick-up ions, from the comet's halo of neutral hydrogen, were found in this solar wind. Sensors on the Vega spacecraft found a variety of plasma waves propagating inside the bow wave. In order to synthesize all the results, a conference on the exploration of Halley's comet will be held this October

  5. Detecting active comets with SDSS

    Energy Technology Data Exchange (ETDEWEB)

    Solontoi, Michael; Ivezic, Zeljko; /Washington U., Seattle, Astron. Dept.; West, Andrew A.; /MIT, MKI; Claire, Mark; /Washington U., Seattle, Astron. Dept.; Juric, Mario; /Princeton U. Observ.; Becker, Andrew; Jones, Lynne; /Washington U., Seattle, Astron. Dept.; Hall, Patrick B.; /York U., Canada; Kent, Steve; /Fermilab; Lupton, Robert H.; /Princeton U. Observ.; Quinn, Tom; /Washington U., Seattle, Astron. Dept. /Princeton U. Observ.


    Using a sample of serendipitously discovered active comets in the Sloan Digital Sky Survey (SDSS), we develop well-controlled selection criteria for greatly increasing the efficiency of comet identification in the SDSS catalogs. After follow-up visual inspection of images to reject remaining false positives, the total sample of SDSS comets presented here contains 19 objects, roughly one comet per 10 million other SDSS objects. The good understanding of selection effects allows a study of the population statistics, and we estimate the apparent magnitude distribution to r {approx} 18, the ecliptic latitude distribution, and the comet distribution in SDSS color space. The most surprising results are the extremely narrow range of colors for comets in our sample (e.g. root-mean-square scatter of only {approx}0.06 mag for the g-r color), and the similarity of comet colors to those of jovian Trojans. We discuss the relevance of our results for upcoming deep multi-epoch optical surveys such as the Dark Energy Survey, Pan-STARRS, and the Large Synoptic Survey Telescope (LSST), and estimate that LSST may produce a sample of about 10,000 comets over its 10-year lifetime.

  6. Where Do Comets Come From? (United States)

    Van Flandern, Tom


    Proposes a new origin for comets in the solar system, namely, that comets originated in the breakup of a body orbiting the sun in or near the present location of the asteroid belt in the relatively recent past. Predictions related to the theory are discussed. (Author/JN)

  7. Comet or Asteroid? (United States)


    When is a minor object in the solar system a comet? And when is it an asteroid? Until recently, there was little doubt. Any object that was found to display a tail or appeared diffuse was a comet of ice and dust grains, and any that didn't, was an asteroid of solid rock. Moreover, comets normally move in rather elongated orbits, while most asteroids follow near-circular orbits close to the main plane of the solar system in which the major planets move. However, astronomers have recently discovered some `intermediate' objects which seem to possess properties that are typical for both categories. For instance, a strange object (P/1996 N2 - Elst-Pizarro) was found last year at ESO ( ESO Press Photo 36/96 ) which showed a cometary tail, while moving in a typical asteroidal orbit. At about the same time, American scientists found another (1996 PW) that moved in a very elongated comet-type orbit but was completely devoid of a tail. Now, a group of European scientists, by means of observations carried out at the ESO La Silla observatory, have found yet another object that at first appeared to be one more comet/asteroid example. However, continued and more detailed observations aimed at revealing its true nature have shown that it is most probably a comet . Consequently, it has received the provisional cometary designation P/1997 T3 . The Uppsala-DLR Trojan Survey Some time ago, Claes-Ingvar Lagerkvist (Astronomical Observatory, Uppsala, Sweden), in collaboration with Gerhard Hahn, Stefano Mottola, Magnus Lundström and Uri Carsenty (DLR, Institute of Planetary Exploration, Berlin, Germany), started to study the distribution of asteroids near Jupiter. They were particularly interested in those that move in orbits similar to that of Jupiter and which are located `ahead' of Jupiter in the so-called `Jovian L4 Lagrangian point'. Together with those `behind' Jupiter, these asteroids have been given the names of Greek and Trojan Heroes who participated in the famous Trojan war

  8. Multiwavelength Observations of Recent Comets (United States)

    Milam, Stefanie N.; Charnley, Steven B.; Gicquel, Adeline; Cordiner, Martin; Kuan, Yi-Jehng; Chuang, Yo-Ling; Villanueva, Geronimo; DiSanti, Michael A.; Bonev, Boncho P.; Remijan, Anthony J.; hide


    Comets provide important clues to the physical and chemical processes that occurred during the formation and early evolution of the Solar System, and could also have been important for initiating prebiotic chemistry on the early Earth. Comets are comprised of molecular ices, that may be pristine inter-stellar remnants of Solar System formation, along with high-temperature crystalline silicate dust that is indicative of a more thermally varied history in the protosolar nebula. Comparing abundances of cometary parent volatiles, and isotopic fractionation ratios, to those found in the interstellar medium, in disks around young stars, and between cometary families, is vital to understanding planetary system formation and the processing history experienced by organic matter in the so-called interstellar-comet connection. We will present a comparison of molecular abundances in these comets to those observed in others, supporting a long-term effort of building a comet taxonomy based on composition.

  9. Asteroids, Comets, Meteors 2014 (United States)

    Muinonen, K.; Penttilä, A.; Granvik, M.; Virkki, A.; Fedorets, G.; Wilkman, O.; Kohout, T.


    Asteroids, Comets, Meteors focuses on the research of small Solar System bodies. Small bodies are the key to understanding the formation and evolution of the Solar System, carrying signals from pre-solar times. Understanding the evolution of the Solar System helps unveil the evolution of extrasolar planetary systems. Societally, small bodies will be important future resources of minerals. The near-Earth population of small bodies continues to pose an impact hazard, whether it be small pieces of falling meteorites or larger asteroids or cometary nuclei capable of causing global environmental effects. The conference series entitled ''Asteroids, Comets, Meteors'' constitutes the leading international series in the field of small Solar System bodies. The first three conferences took place in Uppsala, Sweden in 1983, 1985, and 1989. The conference is now returning to Nordic countries after a quarter of a century. After the Uppsala conferences, the conference has taken place in Flagstaff, Arizona, U.S.A. in 1991, Belgirate, Italy in 1993, Paris, France in 1996, Ithaca, New York, U.S.A. in 1999, in Berlin, Germany in 2002, in Rio de Janeiro, Brazil in 2005, in Baltimore, Maryland, U.S.A. in 2008, and in Niigata, Japan in 2012. ACM in Helsinki, Finland in 2014 will be the 12th conference in the series.

  10. Standardizing electrophoresis conditions: how to eliminate a major source of error in the comet assay.

    Directory of Open Access Journals (Sweden)

    Gunnar Brunborg


    Full Text Available In the alkaline comet assay, cells are embedded in agarose, lysed, and then subjected to further processing including electrophoresis at high pH (>13. We observed very large variations of mean comet tail lengths of cell samples from the same population when spread on a glass or plastic substrate and subjected to electrophoresis. These variations might be cancelled out if comets are scored randomly over a large surface, or if all the comets are scored. The mean tail length may then be representative of the population, although its standard error is large. However, the scoring process often involves selection of 50 – 100 comets in areas selected in an unsystematic way from a large gel on a glass slide. When using our 96-sample minigel format (1, neighbouring sample variations are easily detected. We have used this system to study the cause of the comet assay variations during electrophoresis and we have defined experimental conditions which reduce the variations to a minimum. We studied the importance of various physical parameters during electrophoresis: (i voltage; (ii duration of electrophoresis; (iii electric current; (iv temperature; and (v agarose concentration. We observed that the voltage (V/cm varied substantially during electrophoresis, even within a few millimetres of distance between gel samples. Not unexpectedly, both the potential ( V/cm and the time were linearly related to the mean comet tail, whereas the current was not. By measuring the local voltage with microelectrodes a few millimetres apart, we observed substantial local variations in V/cm, and they increased with time. This explains the large variations in neighbouring sample comet tails of 25% or more. By introducing simple technology (circulation of the solution during electrophoresis, and temperature control, these variations in mean comet tail were largely abolished, as were the V/cm variations. Circulation was shown to be particularly important and optimal conditions

  11. Testing the genotoxicity of some perfumes with high diethylphthalate (DEP levels using comet assay

    Directory of Open Access Journals (Sweden)

    Iman Al-Saleh


    Full Text Available The presence of phthalates in perfumes has gained recently some attention since these chemicals are added sometimes intentionally as a fixative. Our previous study tested 47 branded perfumes sold in Saudi market and found 68% of tested samples had diethylphthalate (DEP, above reported threshold limit of 1 ppm. Of these phthalates, DEP was found to have the highest mean value (1621.63 ppm. These results enticed us to test the potential genotoxicity of 7 brands in which their DEP contents were in the range of 1.06 ppm to 23649.3 ppm in human TK-6 cells using single cell gel electrophoresis assay (comet assay. Cells were exposed to 400 µl perfume for 2 hrs, at room temperature. Tail moment was (TM used as a metric measure for DNA damage and 25 cells per sample were determined by image analysis software. Four perfumes with DEP above 40 ppm produced significant DNA damage in TK-6 cells with TM of 54.27 ± 1.04, n=500 compare to the other 3 perfumes with DEP < 2 ppm (21.94 ± 1.09, n=300, untreated cells (2.99 ± 0.17, n=250 and cells induced with ethanol (33.76 ± 1.4, n=75 or methanol (23.82 ± 1.84, n=100 which are the vehicles used in perfume's preparations. The results suggest that DEP in perfumes might be one of the ingredients that provoked DNA damage; however, further investigation is required confirming our observation.

  12. Application of DNA comet assay for detection of radiation treatment of grams and pulses. (United States)

    Khan, Hasan M; Khan, Ashfaq A; Khan, Sanaullah


    Several types of whole pulses (green lentils, red lentils, yellow lentils, chickpeas, green peas, cowpeas and yellow peas) and grams (black grams, red grams and white grams) have been investigated for the identification of radiation treatment using microgel electrophoresis of single cells (DNA comet assay). Pulses and grams were exposed to the radiation doses of 0.5, 1.0 and 5 kGy covering the legalized commercial dose range for protection from insect/pest infestations. All irradiated samples showed comet like stretching of fragmented DNA toward anode, which is expected for irradiated samples. Unirradiated samples showed many intact cells/nuclei in form of round stains or with short faint tails, which is typical for unirradiated food samples. The study shows that DNA comet assay can be used as a rapid, inexpensive and highly effective screening test for the detection of radiation treatment of foods, like pulses and grams.

  13. EPOXI at comet Hartley 2. (United States)

    A'Hearn, Michael F; Belton, Michael J S; Delamere, W Alan; Feaga, Lori M; Hampton, Donald; Kissel, Jochen; Klaasen, Kenneth P; McFadden, Lucy A; Meech, Karen J; Melosh, H Jay; Schultz, Peter H; Sunshine, Jessica M; Thomas, Peter C; Veverka, Joseph; Wellnitz, Dennis D; Yeomans, Donald K; Besse, Sebastien; Bodewits, Dennis; Bowling, Timothy J; Carcich, Brian T; Collins, Steven M; Farnham, Tony L; Groussin, Olivier; Hermalyn, Brendan; Kelley, Michael S; Kelley, Michael S; Li, Jian-Yang; Lindler, Don J; Lisse, Carey M; McLaughlin, Stephanie A; Merlin, Frédéric; Protopapa, Silvia; Richardson, James E; Williams, Jade L


    Understanding how comets work--what drives their activity--is crucial to the use of comets in studying the early solar system. EPOXI (Extrasolar Planet Observation and Deep Impact Extended Investigation) flew past comet 103P/Hartley 2, one with an unusually small but very active nucleus, taking both images and spectra. Unlike large, relatively inactive nuclei, this nucleus is outgassing primarily because of CO(2), which drags chunks of ice out of the nucleus. It also shows substantial differences in the relative abundance of volatiles from various parts of the nucleus.

  14. Atomic force microscopy for the examination of single cell rheology. (United States)

    Okajima, Takaharu


    Rheological properties of living cells play important roles in regulating their various biological functions. Therefore, measuring cell rheology is crucial for not only elucidating the relationship between the cell mechanics and functions, but also mechanical diagnosis of single cells. Atomic force microscopy (AFM) is becoming a useful technique for single cell diagnosis because it allows us to measure the rheological properties of adherent cells at any region on the surface without any modifications. In this review, we summarize AFM techniques for examining single cell rheology in frequency and time domains. Recent applications of AFM for investigating the statistical analysis of single cell rheology in comparison to other micro-rheological techniques are reviewed, and we discuss what specificity and universality of cell rheology are extracted using AFM.

  15. Functional Insights into Sponge Microbiology by Single Cell Genomics

    KAUST Repository

    Hentschel, Ute


    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

  16. New Array Approaches to Explore Single Cells Genomes (United States)

    Vanneste, Evelyne; Bittman, Lilach; Van der Aa, Niels; Voet, Thierry; Vermeesch, Joris Robert


    Microarray analysis enables the genome-wide detection of copy number variations and the investigation of chromosomal instability. Whereas array techniques have been well established for the analysis of unamplified DNA derived from many cells, it has been more challenging to enable the accurate analysis of single cell genomes. In this review, we provide an overview of single cell DNA amplification techniques, the different array approaches, and discuss their potential applications to study human embryos. PMID:22509179

  17. Recent Trends on Micro/Nanofluidic Single Cell Electroporation

    Directory of Open Access Journals (Sweden)

    Tuhin Subhra Santra


    Full Text Available The behaviors of cell to cell or cell to environment with their organelles and their intracellular physical or biochemical effects are still not fully understood. Analyzing millions of cells together cannot provide detailed information, such as cell proliferation, differentiation or different responses to external stimuli and intracellular reaction. Thus, single cell level research is becoming a pioneering research area that unveils the interaction details in high temporal and spatial resolution among cells. To analyze the cellular function, single cell electroporation can be conducted by employing a miniaturized device, whose dimension should be similar to that of a single cell. Micro/nanofluidic devices can fulfill this requirement for single cell electroporation. This device is not only useful for cell lysis, cell to cell fusion or separation, insertion of drug, DNA and antibodies inside single cell, but also it can control biochemical, electrical and mechanical parameters using electroporation technique. This device provides better performance such as high transfection efficiency, high cell viability, lower Joule heating effect, less sample contamination, lower toxicity during electroporation experiment when compared to bulk electroporation process. In addition, single organelles within a cell can be analyzed selectively by reducing the electrode size and gap at nanoscale level. This advanced technique can deliver (in/out biomolecules precisely through a small membrane area (micro to nanoscale area of the single cell, known as localized single cell membrane electroporation (LSCMEP. These articles emphasize the recent progress in micro/nanofluidic single cell electroporation, which is potentially beneficial for high-efficient therapeutic and delivery applications or understanding cell to cell interaction.

  18. High-throughput single-cell manipulation in brain tissue.

    Directory of Open Access Journals (Sweden)

    Joseph D Steinmeyer

    Full Text Available The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.

  19. COnsortium of METabolomics Studies (COMETS) (United States)

    The COnsortium of METabolomics Studies (COMETS) is an extramural-intramural partnership that promotes collaboration among prospective cohort studies that follow participants for a range of outcomes and perform metabolomic profiling of individuals.

  20. Evaluation of DNA Damage in Common Carp (Cyprinus carpio L. by Comet Assay for Determination of Possible Pollution in Lake Mogan (Ankara

    Directory of Open Access Journals (Sweden)

    İsmet Çok


    Full Text Available Contamination of the aquatic environment with various concentrations of pollutants results in unexpected threats to humans and wildlife. The consequences of exposure and metabolism of pollutants/xenobiotics, especially carcinogens and mutagens, can be suitably assessed by investigating severe events, such as DNA damage; for example, DNA adducts and DNA strand breaks. One of the commonly used techniques to detect DNA damage in aquatic organisms is single-cell gel electrophoresis (comet assay. This study was carried out using Cyprinus carpio in order to identify the possible pollution in Lake Mogan, near Ankara, Turkey, where the city's sewer system and pesticides used in agriculture are believed to be the common causes of pollution. From the comet assay, the tail length (μm, tail intensity (%, and tail moment values of fish caught from Lake Mogan were found to be 31.10 ± 10.39, 7.77 ± 4.51, 1.50 ± 1.48, respectively, whereas for clean reference sites they were found to be 22.80 ± 1.08, 3.47 ± 1.59, 0.40 ± 0.51, respectively. The values are statistically different from each other (p < 0.0001, p < 0.0001, and p < 0.0013, respectively. These results indicate that Lake Mogan may be polluted with substances that have genotoxic effects and constitute an early warning for the lake system. Further detailed research is needed to establish the source of the pollution and the chemicals responsible.

  1. Image analysis driven single-cell analytics for systems microbiology. (United States)

    Balomenos, Athanasios D; Tsakanikas, Panagiotis; Aspridou, Zafiro; Tampakaki, Anastasia P; Koutsoumanis, Konstantinos P; Manolakos, Elias S


    Time-lapse microscopy is an essential tool for capturing and correlating bacterial morphology and gene expression dynamics at single-cell resolution. However state-of-the-art computational methods are limited in terms of the complexity of cell movies that they can analyze and lack of automation. The proposed Bacterial image analysis driven Single Cell Analytics (BaSCA) computational pipeline addresses these limitations thus enabling high throughput systems microbiology. BaSCA can segment and track multiple bacterial colonies and single-cells, as they grow and divide over time (cell segmentation and lineage tree construction) to give rise to dense communities with thousands of interacting cells in the field of view. It combines advanced image processing and machine learning methods to deliver very accurate bacterial cell segmentation and tracking (F-measure over 95%) even when processing images of imperfect quality with several overcrowded colonies in the field of view. In addition, BaSCA extracts on the fly a plethora of single-cell properties, which get organized into a database summarizing the analysis of the cell movie. We present alternative ways to analyze and visually explore the spatiotemporal evolution of single-cell properties in order to understand trends and epigenetic effects across cell generations. The robustness of BaSCA is demonstrated across different imaging modalities and microscopy types. BaSCA can be used to analyze accurately and efficiently cell movies both at a high resolution (single-cell level) and at a large scale (communities with many dense colonies) as needed to shed light on e.g. how bacterial community effects and epigenetic information transfer play a role on important phenomena for human health, such as biofilm formation, persisters' emergence etc. Moreover, it enables studying the role of single-cell stochasticity without losing sight of community effects that may drive it.

  2. Bioinformatics approaches to single-cell analysis in developmental biology. (United States)

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


    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

  3. Tabulation of comet observations. (United States)


    Concerning comets: 1980 IV Černis-Petrauskas, 1985 XVII Hartley-Good, 1985 XIII P/Giacobini-Zinner, 1985 XIX Thiele, 1986 III P/Halley, 1988 I Ichimura, 1988 V Liller, 1988 XV Machholz, 1989 X P/Brorsen-Metcalf, 1989 XIX Okazaki-Levy-Rudenko, 1989 XXII Aarseth-Brewington, 1990 V Austin, 1990 XVII Tsuchiya-Kiuchi, 1990 XX Levy, 1991 I P/Metcalf-Brewington, 1991 XXIV Shoemaker-Levy, 1991l Helin-Lawrence, 1991a1 Shoemaker-Levy, 1991g1 Zanotta-Brewington, 1991h1 Mueller, 1992d Tanaka-Machholz, 1992h Spacewatch, 1992l P/Giclas, 1992m P/Wolf, 1992n P/Faye, 1992p P/Brewington, 1992q Helin-Lawrence, 1992s P/Ciffréo, 1992t P/Swift-Tuttle, 1992u P/Väisälä, 1992x P/Schaumasse, 1992y Shoemaker, 1992a1 Ohshita, 1993a Mueller, 1993d Mueller, 1993e P/Shoemaker-Levy 9, P/Encke, P/Schwassmann-Wachmann 1.

  4. Platforms for Single-Cell Collection and Analysis

    Directory of Open Access Journals (Sweden)

    Lukas Valihrach


    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.

  5. Novel approaches in function-driven single-cell genomics. (United States)

    Doud, Devin F R; Woyke, Tanja


    Deeper sequencing and improved bioinformatics in conjunction with single-cell and metagenomic approaches continue to illuminate undercharacterized environmental microbial communities. This has propelled the 'who is there, and what might they be doing' paradigm to the uncultivated and has already radically changed the topology of the tree of life and provided key insights into the microbial contribution to biogeochemistry. While characterization of 'who' based on marker genes can describe a large fraction of the community, answering 'what are they doing' remains the elusive pinnacle for microbiology. Function-driven single-cell genomics provides a solution by using a function-based screen to subsample complex microbial communities in a targeted manner for the isolation and genome sequencing of single cells. This enables single-cell sequencing to be focused on cells with specific phenotypic or metabolic characteristics of interest. Recovered genomes are conclusively implicated for both encoding and exhibiting the feature of interest, improving downstream annotation and revealing activity levels within that environment. This emerging approach has already improved our understanding of microbial community functioning and facilitated the experimental analysis of uncharacterized gene product space. Here we provide a comprehensive review of strategies that have been applied for function-driven single-cell genomics and the future directions we envision. © FEMS 2017.

  6. The mass disruption of Oort cloud comets. (United States)

    Levison, Harold F; Morbidelli, Alessandro; Dones, Luke; Jedicke, Robert; Wiegert, Paul A; Bottke, William F


    We have calculated the number of dormant, nearly isotropic Oort cloud comets in the solar system by (i) combining orbital distribution models with statistical models of dormant comet discoveries by well-defined surveys and (ii) comparing the model results to observations of a population of dormant comets. Dynamical models that assume that comets are not destroyed predict that we should have discovered approximately 100 times more dormant nearly isotropic comets than are actually seen. Thus, as comets evolve inward from the Oort cloud, the majority of them must physically disrupt.

  7. Review of methods to probe single cell metabolism and bioenergetics. (United States)

    Vasdekis, Andreas E; Stephanopoulos, Gregory


    Single cell investigations have enabled unexpected discoveries, such as the existence of biological noise and phenotypic switching in infection, metabolism and treatment. Herein, we review methods that enable such single cell investigations specific to metabolism and bioenergetics. Firstly, we discuss how to isolate and immobilize individuals from a cell suspension, including both permanent and reversible approaches. We also highlight specific advances in microbiology for its implications in metabolic engineering. Methods for probing single cell physiology and metabolism are subsequently reviewed. The primary focus therein is on dynamic and high-content profiling strategies based on label-free and fluorescence microspectroscopy and microscopy. Non-dynamic approaches, such as mass spectrometry and nuclear magnetic resonance, are also briefly discussed. Published by Elsevier Inc.

  8. Microfluidics-Enabled Enzyme Activity Measurement in Single Cells. (United States)

    Tesauro, Cinzia; Frøhlich, Rikke; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R


    Cellular heterogeneity has presented a significant challenge in the studies of biology. While most of our understanding is based on the analysis of ensemble average, individual cells may process information and respond to perturbations very differently. Presented here is a highly sensitive platform capable of measuring enzymatic activity at the single-cell level. The strategy innovatively combines a rolling circle-enhanced enzyme activity detection (REEAD) assay with droplet microfluidics. The single-molecule sensitivity of REEAD allows highly sensitive detection of enzymatic activities, i.e. at the single catalytic event level, whereas the microfluidics enables isolation of single cells. Further, confined reactions in picoliter-sized droplets significantly improve enzyme extraction from human cells or microorganisms and result in faster reaction kinetics. Taken together, the described protocol is expected to open up new possibilities in the single-cell research, particularly for the elucidation of heterogeneity in a population of cells.

  9. Single-cell sequencing in stem cell biology. (United States)

    Wen, Lu; Tang, Fuchou


    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.

  10. The Use of Evolutionary Approaches to Understand Single Cell Genomes

    Directory of Open Access Journals (Sweden)

    Haiwei eLuo


    Full Text Available The vast majority of environmental bacteria and archaea remain uncultivated, yet their genome sequences are rapidly becoming available through single cell sequencing technologies. Reconstructing metabolism is one common way to make use of genome sequences of ecologically important bacteria, but molecular evolutionary analysis is another approach that, while currently underused, can reveal important insights into the function of these uncultivated microbes in nature. Because genome sequences from single cells are often incomplete, metabolic reconstruction based on genome content can be compromised. However, this problem does not necessarily impede the use of phylogenomic and population genomic approaches that are based on patterns of polymorphisms and substitutions at nucleotide and amino acid sites. These approaches explore how various evolutionary forces act to assemble genetic diversity within and between lineages. In this mini-review, I present examples illustrating the benefits of analyzing single cell genomes using evolutionary approaches.

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

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis


    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.

  12. Single-cell technologies to study the immune system. (United States)

    Proserpio, Valentina; Mahata, Bidesh


    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.

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


    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, 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.

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

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner


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

  15. Comet showers and Nemesis, the death star

    International Nuclear Information System (INIS)

    Hills, J.G.


    The recently proposed hypothesis that the periodic extinctions of terrestrial species are the result of comet showers catalyzed by a hypothetical distant solar companion, Nemesis, a tale of global death by comet bombardment of the earth, is discussed

  16. Identification of radiation treatment of foods using novel technique of 'DNA comet assay'

    International Nuclear Information System (INIS)

    Khan, A.A.; Khan, H.M.; Wasim, M.A.


    Treatment of food using ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests and to extend shelf life thereby contributing to safer and more plentiful food supply. Food control agencies throughout the world need some reliable, simple and rapid methods for the detection foods to ensure free choice of consumer and to enforce labeling. The DNA comet assay offers great potential as a rapid tool to screen irradiated and unirradiated samples of several kinds of foods. In the present study samples of fresh and frozen beef has investigated for the detection of irradiation treatment. The samples were subjected to radiation doses of 0,4.5 and 7 KGy and were stored in freezer before analysis. The cells were extracted into cold PBS solutions, embedded into agarose gel on microscope slides, lysed and eletrophoressed at a voltage of 2V/cm for 2 minutes. The fragmented DNA as a irradiation treatment was stretched in the gel producing the dose dependent comets. These comets were visible using a simple transmission microscope after silver staining. The controlled and irradiation samples of meat were clearly distinguishable on the basis of the stained patterns of DNA in from of round or conical intact cells for unirradiated samples or in from of comets for irradiated samples. It is therefore concluded that DNA comet Assay offers a potential to screen unirradiated and irradiated meat samples. (author)

  17. Detection of radiation treatment of meat by novel techniques of DNA comet assay

    International Nuclear Information System (INIS)

    Khan, A.A.; Khan, H.M.


    Treatment of food to ionizing radiation is being progressively used in many countries to inactivate food pathogens, to eradicate pests and to extend shelf life; thereby contributing to safer and more plentiful food supply. Food control agencies throughout the world need some reliable, simple and rapid methods for detection of irradiated foods to ensure free choice of consumer and to enforce labeling. The DNA comet assay offers great potential as a rapid tool to screen irradiated and unirradiated samples of several kinds of foods. In the present study, frozen beef has been investigated for detection of irradiation treatment. The samples were subjected to radiation doses of 0,4,5 and 7.0 kGy and were stored in freezer before analysis. The cells were extracted into cold PBS solutions, embedded into the agarose gel on microscope slides, lysed and electrophoressed at a voltage of 2v/cm for 2 min. The fragmented DNA as a result of irradiation treatment was stretched in the gel producing the dose dependent comets. These comets were visible using a simple transmission microscope after silver staining. The controlled and irradiated samples of meat were clearly distinguishable on the basis of the stained patterns of DNA in form of round or conical intact cells for unirradiated samples or in form of comets for irradiated samples. It is therefore, concluded that 'DNA Comet Assay' offers a potential to screen unirradiated and irradiated meat samples. (author)


    Energy Technology Data Exchange (ETDEWEB)

    Keane, Jacqueline V.; Kleyna, Jan T.; Riesen, Timm-Emmanuel; Meech, Karen J. [Institute for Astronomy, University of Hawaii, 2680 Woodlawn Drive, Honolulu, HI 96822 (United States); Milam, Stefanie N.; Charnley, Steven B. [Astrochemistry Laboratory, NASA GSFC, MS 690, Greenbelt, MD 20771 (United States); Coulson, Iain M. [Joint Astronomy Center, 660 North Aohoku Place, Hilo, HI 96720 (United States); Sekanina, Zdenek [Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, CA 91109 (United States); Kracht, Rainer, E-mail: [Ostlandring 53, D-25335 Elmshorn, Schleswig-Holstein (Germany)


    We report submillimeter 450 and 850 μ m dust continuum observations for comet C/2012 S1 (ISON) obtained at heliocentric distances 0.31–0.08 au prior to perihelion on 2013 November 28 ( r {sub h} = 0.0125 au). These observations reveal a rapidly varying dust environment in which the dust emission was initially point-like. As ISON approached perihelion, the continuum emission became an elongated dust column spread out over as much as 60″ (>10{sup 5} km) in the anti-solar direction. Deconvolution of the November 28.04 850 μ m image reveals numerous distinct clumps consistent with the catastrophic disruption of comet ISON, producing ∼5.2 × 10{sup 10} kg of submillimeter-sized dust. Orbital computations suggest that the SCUBA-2 emission peak coincides with the comet's residual nucleus.

  19. Catastrophic Disruption of Comet ISON (United States)

    Keane, Jacqueline V.; Milam, Stefanie N.; Coulson, Iain M.; Kleyna, Jan T.; Sekanina, Zdenek; Kracht, Rainer; Riesen, Timm-Emmanuel; Meech, Karen J.; Charnley, Steven B.


    We report submillimeter 450 and 850 microns dust continuum observations for comet C/2012 S1 (ISON) obtained at heliocentric distances 0.31-0.08 au prior to perihelion on 2013 November 28 (rh?=?0.0125 au). These observations reveal a rapidly varying dust environment in which the dust emission was initially point-like. As ISON approached perihelion, the continuum emission became an elongated dust column spread out over as much as 60? (greater than 10(exp 5) km in the anti-solar direction. Deconvolution of the November 28.04 850 microns image reveals numerous distinct clumps consistent with the catastrophic disruption of comet ISON, producing approximately 5.2?×?10(exp 10) kg of submillimeter-sized dust. Orbital computations suggest that the SCUBA-2 emission peak coincides with the comet's residual nucleus.

  20. Hummingbird Comet Nucleus Analysis Mission (United States)

    Kojiro, Daniel; Carle, Glenn C.; Lasher, Larry E.


    Hummingbird is a highly focused scientific mission, proposed to NASA s Discovery Program, designed to address the highest priority questions in cometary science-that of the chemical composition of the cometary nucleus. After rendezvous with the comet, Hummingbird would first methodically image and map the comet, then collect and analyze dust, ice and gases from the cometary atmosphere to enrich characterization of the comet and support landing site selection. Then, like its namesake, Hummingbird would carefully descend to a pre-selected surface site obtaining a high-resolution image, gather a surface material sample, acquire surface temperature and then immediately return to orbit for detailed chemical and elemental analyses followed by a high resolution post-sampling image of the site. Hummingbird s analytical laboratory contains instrumentation for a comprehensive molecular and elemental analysis of the cometary nucleus as well as an innovative surface sample acquisition device.

  1. CO2 Orbital Trends in Comets (United States)

    Kelley, Michael; Feaga, Lori; Bodewits, Dennis; McKay, Adam; Snodgrass, Colin; Wooden, Diane


    Spacecraft missions to comets return a treasure trove of details of their targets, e.g., the Rosetta mission to comet 67P/Churyumov-Gerasimenko, the Deep Impact experiment at comet 9P/Tempel 1, or even the flyby of C/2013 A1 (Siding Spring) at Mars. Yet, missions are rare, the diversity of comets is large, few comets are easily accessible, and comet flybys essentially return snapshots of their target nuclei. Thus, telescopic observations are necessary to place the mission data within the context of each comet's long-term behavior, and to further connect mission results to the comet population as a whole. We propose a large Cycle 11 project to study the long-term activity of past and potential future mission targets, and select bright Oort cloud comets to infer comet nucleus properties, which would otherwise require flyby missions. In the classical comet model, cometary mass loss is driven by the sublimation of water ice. However, recent discoveries suggest that the more volatile CO and CO2 ices are the likely drivers of some comet active regions. Surprisingly, CO2 drove most of the activity of comet Hartley 2 at only 1 AU from the Sun where vigorous water ice sublimation would be expected to dominate. Currently, little is known about the role of CO2 in comet activity because telluric absorptions prohibit monitoring from the ground. In our Cycle 11 project, we will study the CO2 activity of our targets through IRAC photometry. In conjunction with prior observations of CO2 and CO, as well as future data sets (JWST) and ongoing Earth-based projects led by members of our team, we will investigate both long-term activity trends in our target comets, with a particular goal to ascertain the connections between each comet's coma and nucleus.

  2. Infrared techniques for comet observations (United States)

    Hanner, Martha S.; Tokunaga, Alan T.


    The infrared spectral region (1-1000 microns) is important for studies of both molecules and solid grains in comets. Infrared astronomy is in the midst of a technological revolution, with the development of sensitive 2D arrays leading to IR cameras and spectrometers with vastly improved sensitivity and resolution. The Halley campaign gave us tantalizing first glimpses of the comet science possible with this new technology, evidenced, for example, by the many new spectral features detected in the infrared. The techniques of photometry, imaging, and spectroscopy are reviewed in this chapter and their status at the time of the Halley observations is described.

  3. 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 ... that the 50% yeast SCP fed fish had the highest percentage of body protein (55.35%), but with a lower amount of fat at the end of the feeding trial compared to the control.

  4. Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance

    Directory of Open Access Journals (Sweden)

    Felix Schmidt


    Full Text Available Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1 single cell isolation (e.g., by laser-capture microdissection, fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase, and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems. Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients.

  5. Cloning of Plasmodium falciparum by single-cell sorting (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang


    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


    African Journals Online (AJOL)


    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 ...

  7. Production of single cell proterin from brewery spent grains ...

    African Journals Online (AJOL)

    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae obtained from the Federal Institute of Industrial Research Oshtxli was studied by using the extract of spent grains obtained from the International Beer and Beverage Industries, Kacltuia, Nigeria as a substrate in a medium ...

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

    CERN Multimedia

    CERN PhotoLab


    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. Research highlights: microfluidic-enabled single-cell epigenetics. (United States)

    Dhar, Manjima; Khojah, Reem; Tay, Andy; Di Carlo, Dino


    Individual cells are the fundamental unit of life with diverse functions from metabolism to motility. In multicellular organisms, a single genome can give rise to tremendous variability across tissues at the single-cell level due to epigenetic differences in the genes that are expressed. Signals from the local environment or a history of signals can drive these variations, and tissues have many cell types that play separate roles. This epigenetic heterogeneity is of biological importance in normal functions such as tissue morphogenesis and can contribute to development or resistance of cancer, or other disease states. Therefore, an improved understanding of variations at the single cell level are fundamental to understanding biology and developing new approaches to combating disease. Traditional approaches to characterize epigenetic modifications of chromatin or the transcriptome of cells have often focused on blended responses of many cells in a tissue; however, such bulk measures lose spatial and temporal differences that occur from cell to cell, and cannot uncover novel or rare populations of cells. Here we highlight a flurry of recent activity to identify the mRNA profiles from thousands of single-cells as well as chromatin accessibility and histone marks on single to few hundreds of cells. Microfluidics and microfabrication have played a central role in the range of new techniques, and will likely continue to impact their further development towards routine single-cell epigenetic analysis.

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

    African Journals Online (AJOL)



    Mar 13, 2018 ... ABSTRACT: The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage ... as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard techniques. ..... Waste to Wealth- Value Recovery from. Agrofood.

  11. 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 ...

  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. High-dimensional single-cell cancer biology. (United States)

    Irish, Jonathan M; Doxie, Deon B


    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.

  14. 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 ...

  15. Magnetic domain wall conduits for single cell applications

    DEFF Research Database (Denmark)

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


    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 technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation....

  16. Single-cell Analysis of Lambda Immunity Regulation

    DEFF Research Database (Denmark)

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


    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...

  17. Comet Mineralogy as Inferred from Infrared Spectra of Comets (United States)

    Wooden, Diane H.


    For most comets, infrared (IR) spectroscopy (remote sensing) is the method through which we diagnose the mineralogy and size distribution of dust in their comae. The shape and contrast of the IR spectral features depend on the particle size: optically active minerals (absorbing of visible and near-IR solar photons) and submicron solid grains or highly porous (> 90% vacuum) grains primarily contribute to the shapes of the observed resonances. Comet mineralogies typically are determined by fitting thermal emission models of ensembles of discrete mineral grains to observed IR spectral energy distributions. The absorptivities (Q-abs) and scattering efficiencies (Q-scat) of the discrete mineral grains are computed using Mie scattering, Maxwell-Garnet mixing, Discrete Dipole Approximation, and Multi-Layered Sphere codes. These techniques when applied to crystalline minerals, specifically olivine (Mg_x, Fe_1-x)2 Si04, x>0.9, require the use of ellipsoidal shaped particles with elongated axial ratios or hollow spheres to produce the shapes of the resonances observed both from comet comae and laboratory samples. The wavelength positions of the distinct resonances from submicron-radii crystalline silicates, as well as their thermal equilibrium temperatures, constrain the crystalline olivine to have a relatively high Mg-content (x>0.9, or Fo>90). Only resonances computed for submicron Mg-rich crystalline olivine and crystalline orthopyroxene match the observed IR spectral features. However, this has led to the interpretation that micron-radii and larger crystals are absent from comet comae. Furthermore, the mass fraction of silicate crystals is dependent upon whether just the submicron portion of the size distribution is being compared or the submicron crystals compare to the aggregates of porous amorphous silicates that are computationally tractable as porous spheres. We will discuss the Deep Impact results as examples of these challenges to interpreting mid-IR spectra of

  18. Identification of irradiated refrigerated pork with the DNA comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, M.M. E-mail:; Marin-Huachaca, N.S.; Mancini-Filho, J. E-mail:; Delincee, H.; Villavicencio, A.L.C.H. E-mail:


    Food irradiation can contribute to a safer and more plentiful food supply by inactivating pathogens, eradicating pests and by extending shelf-life. Particularly in the case of pork meat, this process could be a useful way to inactivate harmful parasites such as Trichinella and Taenia solium. Ionizing radiation causes damage to the DNA of the cells (e.g. strand breaks), which can be used to detect irradiated food. Microelectrophoresis of single cells ('Comet Assay') is a simple and rapid test for DNA damage and can be used over a wide dose range and for a variety of products. Refrigerated pork meat was irradiated with a {sup 60}Co source, Gammacell 220 (A.E.C.L.) installed in IPEN (Sao Paulo, Brazil). The doses given were 0, 1.5, 3.0 and 4.5 kGy for refrigerated samples. Immediately after irradiation the samples were returned to the refrigerator (6 deg. C). Samples were kept in the refrigerator after irradiation. Pork meat was analyzed 1, 8 and 10 days after irradiation using the DNA 'Comet Assay'. This method showed to be an inexpensive and rapid technique for qualitative detection of irradiation treatment.

  19. The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives

    Directory of Open Access Journals (Sweden)

    Joaquin ede Lapuente


    Full Text Available Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii To review the development and modifications of the protocols used and the cell types (or tissues used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii To highlight correlations with other

  20. The COMET Sleep Research Platform. (United States)

    Nichols, Deborah A; DeSalvo, Steven; Miller, Richard A; Jónsson, Darrell; Griffin, Kara S; Hyde, Pamela R; Walsh, James K; Kushida, Clete A


    The Comparative Outcomes Management with Electronic Data Technology (COMET) platform is extensible and designed for facilitating multicenter electronic clinical research. Our research goals were the following: (1) to conduct a comparative effectiveness trial (CET) for two obstructive sleep apnea treatments-positive airway pressure versus oral appliance therapy; and (2) to establish a new electronic network infrastructure that would support this study and other clinical research studies. The COMET platform was created to satisfy the needs of CET with a focus on creating a platform that provides comprehensive toolsets, multisite collaboration, and end-to-end data management. The platform also provides medical researchers the ability to visualize and interpret data using business intelligence (BI) tools. COMET is a research platform that is scalable and extensible, and which, in a future version, can accommodate big data sets and enable efficient and effective research across multiple studies and medical specialties. The COMET platform components were designed for an eventual move to a cloud computing infrastructure that enhances sustainability, overall cost effectiveness, and return on investment.

  1. Comet C/2001 J1

    Czech Academy of Sciences Publication Activity Database

    Pravec, Petr; Helin, E.; Lawrence, K.; Kotková, Lenka; Tichá, J.; Tichý, M.

    č. 7623 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  2. The Comet Assay and its applications in the field of ecotoxicology: a mature tool that continues to expand its perspectives (United States)

    de Lapuente, Joaquín; Lourenço, Joana; Mendo, Sónia A.; Borràs, Miquel; Martins, Marta G.; Costa, Pedro M.; Pacheco, Mário


    Since Singh and colleagues, in 1988, launched to the scientific community the alkaline Single Cell Gel Electrophoresis (SCGE) protocol, or Comet Assay, its uses and applications has been increasing. The thematic areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. It has been applied to a wide range of experimental models: bacteria, fungi, cells culture, arthropods, fishes, amphibians, reptiles, mammals, and humans. This document is intended to be a comprehensive review of what has been published to date on the field of ecotoxicology, aiming at the following main aspects: (i) to show the most relevant experimental models used as bioindicators both in the laboratory and in the field. Fishes are clearly the most adopted group, reflecting their popularity as bioindicator models, as well as a primary concern over the aquatic environment health. Amphibians are among the most sensitive organisms to environmental changes, mainly due to an early aquatic-dependent development stage and a highly permeable skin. Moreover, in the terrestrial approach, earthworms, plants or mammalians are excellent organisms to be used as experimental models for genotoxic evaluation of pollutants, complex mix of pollutants and chemicals, in both laboratory and natural environment. (ii) To review the development and modifications of the protocols used and the cell types (or tissues) used. The most recent developments concern the adoption of the enzyme linked assay (digestion with lesion-specific repair endonucleases) and prediction of the ability to repair of oxidative DNA damage, which is becoming a widespread approach, albeit challenging. For practical/technical reasons, blood is the most common choice but tissues/cells like gills, sperm cells, early larval stages, coelomocytes, liver or kidney have been also used. (iii) To highlight correlations with other biomarkers

  3. Modeling Formaldehyde Emission in Comets (United States)

    Disanti, M. A.; Reuter, D. C.; Bonev, B. P.; Mumma, M. J.; Villanueva, G. L.

    Modeling fluorescent emission from monomeric formaldehyde (H2CO) forms an integral part of our overall comprehensive program of measuring the volatile composition of comets through high-resolution (RP ~ 25,000) infrared spectroscopy using CSHELL at the IRTF and NIRSPEC at Keck II. The H2CO spectra contain lines from both the nu1 (symmetric CH2 stretch) and nu5 (asymmetric CH2 stretch) bands near 3.6 microns. We have acquired high-quality spectra of twelve Oort cloud comets, and at least six of these show clear emission from H2CO. We also detected H2CO with NIRSPEC in one Jupiter Family comet, 9P/Tempel 1, during Deep Impact observations. Our H2CO model, originally developed to interpret low-resolution spectra of comets Halley and Wilson (Reuter et al. 1989 Ap J 341:1045), predicts individual line intensities (g-factors) as a function of rotational temperature for approximately 1300 lines having energies up to approximately 400 cm^-1 above the ground state. Recently, it was validated through comparison with CSHELL spectra of C/2002 T7 (LINEAR), where newly developed analyses were applied to obtain robust determinations of both the rotational temperature and abundance of H2CO (DiSanti et al. 2006 Ap J 650:470). We are currently in the process of extending the model to higher rotational energy (i.e., higher rotational quantum number) in an attempt to improve the fit to high-J lines in our spectra of C/T7 and other comets. Results will be presented, and implications discussed.Modeling fluorescent emission from monomeric formaldehyde (H2CO) forms an integral part of our overall comprehensive program of measuring the volatile composition of comets through high-resolution (RP ~ 25,000) infrared spectroscopy using CSHELL at the IRTF and NIRSPEC at Keck II. The H2CO spectra contain lines from both the nu1 (symmetric CH2 stretch) and nu5 (asymmetric CH2 stretch) bands near 3.6 microns. We have acquired high-quality spectra of twelve Oort cloud comets, and at least six of

  4. New library construction method for single-cell genomes.

    Directory of Open Access Journals (Sweden)

    Larry Xi

    Full Text Available A central challenge in sequencing single-cell genomes is the accurate determination of point mutations, phasing of these mutations, and identifying copy number variations with few assumptions. Ideally, this is accomplished under as low sequencing coverage as possible. Here we report our attempt to meet these goals with a novel library construction and library amplification methodology. In our approach, single-cell genomic DNA is first fragmented with saturated transposition to make a primary library that uniformly covers the whole genome by short fragments. The library is then amplified by a carefully optimized PCR protocol in a uniform and synchronized fashion for next-generation sequencing. Each step of the protocol can be quantitatively characterized. Our shallow sequencing data show that the library is tightly distributed and is useful for the determination of copy number variations.

  5. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno


    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.

  6. Single cell analysis contemporary research and clinical applications

    CERN Document Server

    Cossarizza, Andrea


    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.

  7. Single-cell magnetic imaging using a quantum diamond microscope. (United States)

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


    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.


    African Journals Online (AJOL)

    60. N.N. OSADEBE and J.C. EZEH. Figure 1 shows one of the cross sections of a single-cell mono symmetric thin-walled closed column under consideration. Using. Lagrange's principle, Vlasov [6] expressed the displacements in the longitudinal and transverse directions, u(x, s) and v(x, s) of a thin-walled closed structure in ...

  9. Single cell magnetic imaging using a quantum diamond microscope (United States)

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


    We apply a quantum diamond microscope to 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 two orders of magnitude larger field of view (~1 mm2) than previous NV imaging technologies, enabling practical applications. To illustrate, we quantify cancer biomarkers expressed by rare tumor cells in a large population of healthy cells. PMID:26098019

  10. Mie scatter corrections in single cell infrared microspectroscopy. (United States)

    Konevskikh, Tatiana; Lukacs, Rozalia; Blümel, Reinhold; Ponossov, Arkadi; Kohler, Achim


    Strong Mie scattering signatures hamper the chemical interpretation and multivariate analysis of the infrared microscopy spectra of single cells and tissues. During recent years, several numerical Mie scatter correction algorithms for the infrared spectroscopy of single cells have been published. In the paper at hand, we critically reviewed existing algorithms for the correction of Mie scattering and suggest improvements. We developed an iterative algorithm based on Extended Multiplicative Scatter Correction (EMSC), for the retrieval of pure absorbance spectra from highly distorted infrared spectra of single cells. The new algorithm uses the van de Hulst approximation formula for the extinction efficiency employing a complex refractive index. The iterative algorithm involves the establishment of an EMSC meta-model. While existing iterative algorithms for the correction of resonant Mie scattering employ three independent parameters for establishing a meta-model, we could decrease the number of parameters from three to two independent parameters, which reduced the calculation time for the Mie scattering curves for the iterative EMSC meta-model by a factor of 10. Moreover, by employing the Hilbert transform for evaluating the Kramers-Kronig relations based on a FFT algorithm in Matlab, we further improved the speed of the algorithm by a factor of 100. For testing the algorithm we simulate distorted apparent absorbance spectra by utilizing the exact theory for the scattering of infrared light at absorbing spheres, taking into account the high numerical aperture of infrared microscopes employed for the analysis of single cells and tissues. In addition, the algorithm was applied to measured absorbance spectra of single lung cancer cells.

  11. Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity. (United States)

    Angermueller, Christof; Clark, Stephen J; Lee, Heather J; Macaulay, Iain C; Teng, Mabel J; Hu, Tim Xiaoming; Krueger, Felix; Smallwood, Sebastien; Ponting, Chris P; Voet, Thierry; Kelsey, Gavin; Stegle, Oliver; Reik, Wolf


    We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

  12. Femtosecond laser fabrication of optofluidic devices for single cell manipulation

    Directory of Open Access Journals (Sweden)

    Bragheri Francesca


    Full Text Available In this work we fabricate and validate two optofludic devices for the manipulation and analysis of single cells. The chips are fabricated by femtosecond laser micromachining exploiting the 3D capabilities of the technique and the inherent perfect alignment between microfluidic channels and optical networks. Both devices have been validated by probing the mechanical properties of different cancer cell lines, which are expected to show different elasticity because of their different metastatic potential.

  13. Bioanalytical tools for single-cell study of exocytosis. (United States)

    Ge, Shencheng; Koseoglu, Secil; Haynes, Christy L


    Regulated exocytosis is a fundamental biological process used to deliver chemical messengers for cell-cell communication via membrane fusion and content secretion. A plethora of cell types employ this chemical-based communication to achieve crucial functions in many biological systems. Neurons in the brain and platelets in the circulatory system are representative examples utilizing exocytosis for neurotransmission and blood clotting. Single-cell studies of regulated exocytosis in the past several decades have greatly expanded our knowledge of this critical process, from vesicle/granule transport and docking at the early stages of exocytosis to membrane fusion and to eventual chemical messenger secretion. Herein, four main approaches that have been widely used to study single-cell exocytosis will be highlighted, including total internal reflection fluorescence microscopy, capillary electrophoresis, single-cell mass spectrometry, and microelectrochemistry. These techniques are arranged in the order following the route of a vesicle/granule destined for secretion. Within each section, the basic principles and experimental strategies are reviewed and representative examples are given revealing critical spatial, temporal, and chemical information of a secretory vesicle/granule at different stages of its lifetime. Lastly, an analytical chemist's perspective on potential future developments in this exciting field is discussed.

  14. 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


    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.

  15. Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles. (United States)

    Eustaquio, Trisha; Leary, James F


    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.

  16. Clustering Single-Cell Expression Data Using Random Forest Graphs. (United States)

    Pouyan, Maziyar Baran; Nourani, Mehrdad


    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.

  17. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei


    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.

  18. Potentials of single-cell biology in identification and validation of disease biomarkers. (United States)

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


    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.

  19. Thermoresponsive Gels


    Taylor, M. Joan; Tomlins, P.; Sahota, T. S.


    An invited review and relates to the responsive gel used in the "artificial pancreas" work og INsmart, DMU. This article is an Open Access journal. Thermoresponsive gelling materials constructed from natural and synthetic polymers can be used to provide triggered action and therefore customised products such as drug delivery and regenerative medicine types as well as for other industries. Some materials give Arrhenius-type viscosity changes based on coil to globule transitions. Others ...

  20. ISO's analysis of Comet Hale-Bopp (United States)


    The European Space Agency's Infrared Space Observatory ISO inspected Comet Hall-Bopp during the spring and autumn of 1996. The need to keep ISO's telescope extremely cold restricts the spacecraft's pointing in relation to the Sun and the Earth and it ruled out observations at other times. The analyses of the 1996 observations are not yet complete, but already they give new insight into the nature of comets. Comet Hale-Bopp is believed to be a large comet with a nucleus up to 40 kilometres wide. It was discovered in July 1995 by two American astronomers working independently, Alan Hale and Thomas Bopp. At that time, the comet was a billion kilometres away from the Sun, but 200 times brighter than Halley's Comet was, when at a comparable distance. Comet Hale-Bopp will make its closest approach to the Earth on 22 March, and its closest approach to the Sun (perihelion) on 1 April 1997. Some scientific results from ISO The discovery of Comet Hale-Bopp occurred before ISO's launch in November 1995. When first observed by ISO in March and April 1996, the comet was still 700 million kilometres from the Sun, and almost as far from the Earth and ISO. With its privileged view of infrared wavebands inaccessible from the Earth's surface, ISO's photometer ISOPHOT discovered that carbon dioxide was an important constituent of the comet's emissions of vapour.ISOPHOT measured the temperature of the dust cloud around Comet Hale-Bopp. In March 1996, when the comet was still more than 700 million kilometres from the Sun, the dust cloud was at minus 120 degrees C. When ISOPHOT made similar observations in October 1996, the comet was 420 million kilometres from the Sun, and the dust cloud had warmed to about minus 50 degrees C. Intensive observations of Comet Hale-Bopp were also made by ISO's Short-Wave Spectrometer SWS, the Long-Wave Spectrometer LWS, and the ISOPHOT spectrometer PHOT-S. Results are due for publication at the end of March. They will give details about the composition

  1. Comet Halley: nucleus and jets

    International Nuclear Information System (INIS)

    Sagdeev, R.Z.; Avanesov, G.A.; Barinov, I.V.


    The VEGA-1 and VEGA-2 spacecrafts made their closest approach to Comet Halley on 6 and 9 March, respectively. In this paper results of the onboard imaging experiment are discussed. The nucleus of the comet was clearly identifyable as an irregularly shaped object with overall dimensions of (16+-1)x(8+-1)x(8+-1) km. The nucleus rotates around its axis which is nearly perpendicular to the orbital plane, with a period of 53+-2 hours. Its albedo is only 0.04+-002. Most of the jet features observed during the second fly-by were spatially reconstructed. These sources form a quasi-linear structure on the surface. The dust above the surface is shown to be optically thin except certain specific dust jets. Brightness features on the surface are clearly seen. Correlating the data with other measurements it is concluded that the dirty snow-ball model probably has to be revised. (author)

  2. Rationalization of Comet Halley's periods (United States)

    Belton, Michael J. S.


    The sense of long axis orientation of Comet Halley during the Vega 1 encounter must be reversed from that deduced by Sagdeev et al. (1986) in order to harmonize the comet nucleus' Vega/Giotto-observed orientations with periodicities extracted from time-series brightness data. It is also demonstrated that Vega/Giotto observations can be satisfied by either a 2.2- or 3.7-day long-axis free precession period. A novel Fourier algorithm is used to reanalyze five independent data sets; strong evidence is adduced for periods harmonically related to a 7.4-day period. The preferred candidate models for Halley's nuclear rotation are characterized by a long-axis precession period of 3.7 days.

  3. Solar wind stagnation near comets

    International Nuclear Information System (INIS)

    Galeev, A.A.; Cravens, T.E.; Gombosi, T.I.


    The nature of the solar wind flow near comets is examined analytically. In particular, the typical values for the stagnation pressure and magnetic barrier strength are estimated, taking into account the magnetic field line tension and the charge exchange cooling of the mass loaded solar wind. Knowledge of the strength of the magnetic barrier is required in order to determine the location of the contact discontinuity which separates the contaminated solar wind plasma and the outflowing plasma of the cometary ionosphere. (author)

  4. Comet coma sample return instrument (United States)

    Albee, A. L.; Brownlee, Don E.; Burnett, Donald S.; Tsou, Peter; Uesugi, K. T.


    The sample collection technology and instrument concept for the Sample of Comet Coma Earth Return Mission (SOCCER) are described. The scientific goals of this Flyby Sample Return are to return to coma dust and volatile samples from a known comet source, which will permit accurate elemental and isotopic measurements for thousands of individual solid particles and volatiles, detailed analysis of the dust structure, morphology, and mineralogy of the intact samples, and identification of the biogenic elements or compounds in the solid and volatile samples. Having these intact samples, morphologic, petrographic, and phase structural features can be determined. Information on dust particle size, shape, and density can be ascertained by analyzing penetration holes and tracks in the capture medium. Time and spatial data of dust capture will provide understanding of the flux dynamics of the coma and the jets. Additional information will include the identification of cosmic ray tracks in the cometary grains, which can provide a particle's process history and perhaps even the age of the comet. The measurements will be made with the same equipment used for studying micrometeorites for decades past; hence, the results can be directly compared without extrapolation or modification. The data will provide a powerful and direct technique for comparing the cometary samples with all known types of meteorites and interplanetary dust. This sample collection system will provide the first sample return from a specifically identified primitive body and will allow, for the first time, a direct method of matching meteoritic materials captured on Earth with known parent bodies.

  5. Halley comet, implication on the origin

    International Nuclear Information System (INIS)

    Festou, M.C.


    One will first give a rapid description of the different parts that compose a comet coma. Then one will describe the spectrum of comets from the UV to the IR regions with special emphasis on how information relative to the physico-chemistry of comet atmospheres can be retrieved. Our basic knowledge about the composition of comets before 1985 will be summarized and the input of the 1985-86 observing campaign of comet Halley will be shown (in situ, ground-based and space borne observations). One will see then that the chemical composition of comets appears as of today completely compatible with a formation from pre-solar matter that condensed inside the solar system [fr


    Energy Technology Data Exchange (ETDEWEB)

    Perlmutter, S.; Muller, R.A.


    Clustering of cosmic-ray exposure ages of H chondritic meteorites occurs at 7 {+-} 3 and 30 {+-} 6 Myr ago. There is independent evidence that comet storms have occurred at the same times, based on the fossil record of family and genus extinctions, impact craters and glass, and geomagnetic reversals. We suggest that H chondrites were formed by the impact of shower comets on asteroids. The duration of the most recent comet shower was {le} 4 Myr, in agreement with storm theory.

  7. Single Cell Adhesion Assay Using Computer Controlled Micropipette (United States)

    Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint


    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 sub

  8. 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

  9. Cometary Volatiles and the Origin of Comets (United States)

    A'Hearn, Michael F.; Feaga, Lori M.; Keller, H. Uwe; Kawakita, Hideyo; Hampton, Donald L.; Kissel, Jochen; Klaasen, Kenneth P.; McFadden, Lucy A.; Meech, Karen J.; Schultz, Peter H.; hide


    We describe recent results on the CO/C02/H2O composition of comets and compare these with models of the protoplanetary disk. We argue that the cometary observations require reactions on grain surfaces to convert CO to CO2 and also require formation between the CO and CO2 snow lines. This then requires very early mixing of cometesimals in the protoplanetary disk analogous to the mixing described for the asteroid belt by Walsh and Morbidelli. We suggest that most comets formed in the region of the giant planets. the traditional source of the Oort-cloud comets but not of the Jupiter-family comets

  10. Gravity research on plants: use of single cell experimental models

    Directory of Open Access Journals (Sweden)

    Youssef eChebli


    Full Text Available Future space missions and implementation of permanent bases on Moon and Mars will greatly depend on the availability of ambient air and sustainable food supply. Therefore, understanding the effects of altered gravity conditions on plant metabolism and growth is vital for space missions and extra-terrestrial human existence. In this mini-review we summarize how plant cells are thought to perceive changes in magnitude and orientation of the gravity vector. The particular advantages of several single celled model systems for gravity research are explored and an overview over recent advancements and potential use of these systems is provided.

  11. Single-cell sequencing of the small-RNA transcriptome. (United States)

    Faridani, Omid R; Abdullayev, Ilgar; Hagemann-Jensen, Michael; Schell, John P; Lanner, Fredrik; Sandberg, Rickard


    Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.

  12. Single cell array impedance analysis in a microfluidic device (United States)

    Altinagac, Emre; Taskin, Selen; Kizil, Huseyin


    Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are fabricated to capture each cell in a single form and perform impedance analysis. HCT116 (homosapiens colon colorectal carcin) and HEK293 (human embryonic kidney) cells are used in our experiments.

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

    DEFF Research Database (Denmark)

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


    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 preclinical modalities and translation towards clinical application. Conclusions: The development of radiation microbeams has been a valuable tool for the exploration of fundamental radiobiological response mechanisms. The strength of micro-irradiation techniques lies in their ability to deliver precise doses...

  14. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

    DEFF Research Database (Denmark)

    Hou, Yong; Wu, Kui; Shi, Xulian


    BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleoti......BACKGROUND: Single-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate...

  15. Thermoresponsive Gels

    Directory of Open Access Journals (Sweden)

    M. Joan Taylor


    Full Text Available Thermoresponsive gelling materials constructed from natural and synthetic polymers can be used to provide triggered action and therefore customised products such as drug delivery and regenerative medicine types as well as for other industries. Some materials give Arrhenius-type viscosity changes based on coil to globule transitions. Others produce more counterintuitive responses to temperature change because of agglomeration induced by enthalpic or entropic drivers. Extensive covalent crosslinking superimposes complexity of response and the upper and lower critical solution temperatures can translate to critical volume temperatures for these swellable but insoluble gels. Their structure and volume response confer advantages for actuation though they lack robustness. Dynamic covalent bonding has created an intermediate category where shape moulding and self-healing variants are useful for several platforms. Developing synthesis methodology—for example, Reversible Addition Fragmentation chain Transfer (RAFT and Atomic Transfer Radical Polymerisation (ATRP—provides an almost infinite range of materials that can be used for many of these gelling systems. For those that self-assemble into micelle systems that can gel, the upper and lower critical solution temperatures (UCST and LCST are analogous to those for simpler dispersible polymers. However, the tuned hydrophobic-hydrophilic balance plus the introduction of additional pH-sensitivity and, for instance, thermochromic response, open the potential for coupled mechanisms to create complex drug targeting effects at the cellular level.

  16. Genetic biosensors for imaging nitric oxide in single cells. (United States)

    Eroglu, Emrah; Charoensin, Suphachai; Bischof, Helmut; Ramadani, Jeta; Gottschalk, Benjamin; Depaoli, Maria R; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland


    Over the last decades a broad collection of sophisticated fluorescent protein-based probes was engineered with the aim to specifically monitor nitric oxide (NO), one of the most important signaling molecules in biology. Here we report and discuss the characteristics and fields of applications of currently available genetically encoded fluorescent sensors for the detection of NO and its metabolites in different cell types. Because of its radical nature and short half-life, real-time imaging of NO on the level of single cells is challenging. Herein we review state-of-the-art genetically encoded fluorescent sensors for NO and its byproducts such as peroxynitrite, nitrite and nitrate. Such probes enable the real-time visualization of NO signals directly or indirectly on the level of single cells and cellular organelles and, hence, extend our understanding of the spatiotemporal dynamics of NO formation, diffusion and degradation. Here, we discuss the significance of NO detection in individual cells and on subcellular level with genetic biosensors. Currently available genetically encoded fluorescent probes for NO and nitrogen species are critically discussed in order to provide insights in the functionality and applicability of these promising tools. As an outlook we provide ideas for novel approaches for the design and application of improved NO probes and fluorescence imaging protocols. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. 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.


    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

  18. 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


    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.

  19. Single-cell analysis of endothelial morphogenesis in vivo (United States)

    Yu, Jianxin A.; Castranova, Daniel; Pham, Van N.; Weinstein, Brant M.


    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo. PMID:26253401

  20. Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

    Energy Technology Data Exchange (ETDEWEB)

    Lundberg, Derek; Woyke, Tanja; Tringe, Susannah; Dangl, Jeff


    Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the few ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.

  1. Ciliary heterogeneity within a single cell: the Paramecium model. (United States)

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


    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.

  2. Chip based single cell analysis for nanotoxicity assessment. (United States)

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


    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.

  3. Correlated receptor transport processes buffer single-cell heterogeneity. (United States)

    Kallenberger, Stefan M; Unger, Anne L; Legewie, Stefan; Lymperopoulos, Konstantinos; Klingmüller, Ursula; Eils, Roland; Herten, Dirk-Peter


    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.

  4. Single-cell force spectroscopy of pili-mediated adhesion (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.


    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).

  5. Advances of Single-Cell Sequencing Technique in Tumors

    Directory of Open Access Journals (Sweden)

    Ji-feng FENG


    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.

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

    Directory of Open Access Journals (Sweden)

    Stefan M Kallenberger


    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.

  7. DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco Cellular and acellular Comet assays

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš


    Roč. 535, - (2003), s. 187-193 ISSN 1383-5718 R&D Projects: GA ČR GA521/02/0400 Institutional research plan: CEZ:AV0Z5038910 Keywords : Hydrogen peroxide * Single-cell gel electrophoresis * Nicotiana tabacum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.748, year: 2003

  8. Comet Dust After Deep Impact (United States)

    Wooden, Diane H.; Harker, David E.; Woodward, Charles E.


    When the Deep Impact Mission hit Jupiter Family comet 9P/Tempel 1, an ejecta crater was formed and an pocket of volatile gases and ices from 10-30 m below the surface was exposed (A Hearn et aI. 2005). This resulted in a gas geyser that persisted for a few hours (Sugita et al, 2005). The gas geyser pushed dust grains into the coma (Sugita et a1. 2005), as well as ice grains (Schulz et al. 2006). The smaller of the dust grains were submicron in radii (0-25.3 micron), and were primarily composed of highly refractory minerals including amorphous (non-graphitic) carbon, and silicate minerals including amorphous (disordered) olivine (Fe,Mg)2SiO4 and pyroxene (Fe,Mg)SiO3 and crystalline Mg-rich olivine. The smaller grains moved faster, as expected from the size-dependent velocity law produced by gas-drag on grains. The mineralogy evolved with time: progressively larger grains persisted in the near nuclear region, having been imparted with slower velocities, and the mineralogies of these larger grains appeared simpler and without crystals. The smaller 0.2-0.3 micron grains reached the coma in about 1.5 hours (1 arc sec = 740 km), were more diverse in mineralogy than the larger grains and contained crystals, and appeared to travel through the coma together. No smaller grains appeared at larger coma distances later (with slower velocities), implying that if grain fragmentation occurred, it happened within the gas acceleration zone. These results of the high spatial resolution spectroscopy (GEMINI+Michelle: Harker et 4. 2005, 2006; Subaru+COMICS: Sugita et al. 2005) revealed that the grains released from the interior were different from the nominally active areas of this comet by their: (a) crystalline content, (b) smaller size, (c) more diverse mineralogy. The temporal changes in the spectra, recorded by GEMIM+Michelle every 7 minutes, indicated that the dust mineralogy is inhomogeneous and, unexpectedly, the portion of the size distribution dominated by smaller grains has

  9. Spectrophotometry of 25 comets - Post-Halley updates for 17 comets plus new observations for eight additional comets (United States)

    Newburn, Ray L., Jr.; Spinrad, Hyron


    The best possible production figures within the current post-Halley framework and available observations are given for H2O, O(1D), CN, C3, C2 and dust in 25 comets. Of these, the three objects with the smallest mixing ratios of all minor species have moderate to little or no dust and appear 'old'. Comets with large amounts of CN are very dusty, and there is a clear correlation of CN with dust, although comets with little or no dust still have some CN. Thus, CN appears to have at least two sources, dust and one or more parent gases. Also, the C2/CN production ratio changes continuously with heliocentric distance in every comet considered, suggesting that C2 production may be a function of coma density as well as parental abundance. Dust production ranges from essentially zero in Comet Sugano-Saigusa-Fujikawa up to 67,000 kg/s for Halley on March 14, 1986.

  10. Amino Acids from a Comet (United States)

    Cook, Jamie Elisla


    NASA's Stardust spacecraft returned samples from comet 81P/Wild 2 to Earth in January 2006. Examinations of the organic compounds in cometary samples can reveal information about the prebiotic organic inventory present on the early Earth and within the early Solar System, which may have contributed to the origin of life. Preliminary studies of Stardust material revealed the presence of a suite of organic compounds including several amines and amino acids, but the origin of these compounds (cometary- vs. terrestrial contamination) could not be identified. We have recently measured the carbon isotopic ratios of these amino acids to determine their origin, leading to the first detection of a coetary amino acid.

  11. 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.

  12. Ex-dormant comets that come back to life: a search of reactivated comets (United States)

    Quan-Zhi, YE


    Dormant or near-dormant short-period comets can unexpectedly regain the ability to eject dust. In many known cases, the resurrection is short-lived and lasts less than one orbit. However, it is possible that some resurrected comets can remain active in later perihelion passages. Here we conduct a search in the archival images of various facilities to look for these “reactivated” comets. We identify two candidates, 297P/Beshore and 332P/Ikeya-Murakami, both of which were found to be inactive or weakly active in the previous orbit before their discovery. We derive a reactivation rate of 0.007 events per comet per orbit, which implies that typical short-period comets only become temporarily dormant a few times or less. Smaller comets are prone to rotational instability and may undergo temporary dormancy more frequently. Next generation high-cadence surveys may find more reactivation events of these comets.

  13. 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


    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

  14. Comet mission hopes to uncover Earth's origins

    CERN Multimedia

    Henderson, M


    "A European spacecraft that will hunt down a comet in search of clues to the origin of life on Earth will blast off tomorrow from the Kourou spaceport in French Guiana. The Rosetta probe will take 12 years to catch up with Churyumov-Gerasimenko before becoming the first spacecraft to make a soft, controlled landing on a comet's nucleus" (1 page).

  15. Comet assay on mice testicular cells

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar


    of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided...

  16. Meteoroid Streams from Sunskirter Comet Breakup (United States)

    Jenniskens, P. M.


    In its first year of operations, the CAMS project (Cameras for Allsky Meteor Surveillance) has measured 47,000 meteoroid orbits at Earth, including some that pass the Sun as close as 0.008 AU. The population density increases significantly above perihelion distance q = 0.037 AU. Meteoroid streams are known with q about 0.1 AU. The Sun has a profound effect on comets that pass at 0.04-0.16 AU distance, called the sunskirter comets. SOHO and STEREO see families of small comets called the Marsden and Kracht groups. Sunlight is efficiently scattered by small 10-m sized fragments, making those fragments visible even when far from Earth. These comet groups are associated with meteor showers on Earth, in particular the Daytime Arietids and Delta Aquariids. All are related to 96P/Machholz, a highly inclined short-period (5.2 year) Jupiter family comet that comes to within 0.12 AU from the Sun, the smallest perihelion distance known among numbered comets. The proximity of the Sun speeds up the disintegration process, providing us a unique window on this important decay mechanism of Jupiter family comets and creating meteoroid streams. These are not the only sunskirting comets, however. In this presentation, we will present CAMS observations of the complete low-q meteoroid population at Earth and review their association with known parent bodies.

  17. RFP for the Comet Nuclei Tour (CONTOUR)

    DEFF Research Database (Denmark)

    Jørgensen, John Leif; Madsen, Peter Buch; Betto, Maurizio


    This document describes the ASC Star Tracker (performance, functionality, requirements etc.) to The Johns Hopkins University - Applied Physics Laboratory for their Comet Nuclei TOUR (CONTOUR) Program.......This document describes the ASC Star Tracker (performance, functionality, requirements etc.) to The Johns Hopkins University - Applied Physics Laboratory for their Comet Nuclei TOUR (CONTOUR) Program....

  18. The Geology of Comet 19/P Borrelly (United States)

    Britt, D. T.; Boice, D. C; Buratti, B. J.; Hicks, M. D.; Nelson, R. M.; Oberst, J.; Sandel, B. R.; Soderblom, L. A.; Stern, S. A.; Thomas, N.


    The Deep Space One spacecraft flew by Comet 19P/Borrelly on September 22, 2001 and returned a rich array of imagery with resolutions of up to 48 m/pixel. These images provide a window into the surface structure, processes, and geological history of a comet. Additional information is contained in the original extended abstract.

  19. Phobos and Deimos: A Potential Comet Connection (United States)

    Lee, P.


    Phobos and Deimos are similar in size and spectrum to an exceptionally large and spectrally uncommon asteroid among near-Earth objects: 3552 Don Quixote, a D-type NEO that's a comet. The martian moons' potential link to comets is discussed.

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

    Directory of Open Access Journals (Sweden)

    Purnama Sukardi


    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

  1. Comets and How to Observe Them

    CERN Document Server

    Schmude, Richard


    Comets have inspired wonder, excitement and even fear ever since they were first observed. They contain material from early in the life of the Solar System, held in deep-freeze. This makes them key in our understanding of the formation and evolution of many Solar System bodies. Recent ground- and space-based observations have changed much in our understanding of comets. Comets and How to Observe Them gives a summary of our current knowledge and describes how amateur astronomers can contribute to the body of scientific knowledge of comets. This book contains many practical examples of how to construct comet light-curves, measure how fast a comet’s coma expands, and determine the rotation period of the nucleus. All these examples are illustrated with drawings and photographs.

  2. Origin of Short-Perihelion Comets (United States)

    Guliyev, A. S.


    New regularities for short-perihelion comets are found. Distant nodes of cometary orbits of Kreutz family are concentrated in a plane with ascending node 76 and inclination 267 at the distance from 2 up to 3 a.u. and in a very narrow interval of longitudes. There is a correlation dependence between q and cos I concerning the found plane (coefficient of correlation 0.41). Similar results are received regarding to cometary families of Meyer, Kracht and Marsden. Distant nodes of these comets are concentrated close three planes (their parameters are discussed in the article) and at distances 1.4; 0.5; 6 a.u. accordingly. It is concluded that these comet groups were formed as a result of collision of parent bodies with meteoric streams. One more group, consisting of 7 comets is identified. 5 comet pairs are selected among sungrazers.

  3. Correlating Viscoelasticity with Metabolism in Single Cells using Atomic Force Microscopy (United States)

    Caporizzo, Matthew; Roco, Charles; Coll-Ferrer, Carme; Eckmann, David; Composto, Russell


    Variable indentation-rate rheometric analysis by Laplace transform (VIRRAL), is developed to evaluate Dex-Gel drug carriers as biocompatible delivery agents. VIRRAL provides a general platform for the rapid characterization of the health of single cells by viscoelasticity to promote the self-consistent comparison between cells paramount to the development of early diagnosis and treatment of disease. By modelling the frequency dependence of elastic modulus, VIRRAL provides three metrics of cytoplasmic viscoelasticity: low frequency stiffness, high frequency stiffness, and a relaxation time. THP-1 cells are found to exhibit a frequency dependent elastic modulus consistent with the standard linear solid model of viscoelasticity. VIRRAL indicates that dextran-lysozyme drug carriers are biocompatible and deliver concentrated toxic material (rhodamine or silver nanoparticles) to the cytoplasm of THP-1 cells. The signature of cytotoxicity by rhodamine or silver exposure is a frequency independent 2-fold increase in elastic modulus and cytoplasmic viscosity while the cytoskeletal relaxation time remains unchanged independent of cytoplasmic stiffness. This is consistent with the known toxic mechanism of silver nanoparticles, where mitochondrial injury leads to ATP depletion and metabolic stress causes a decrease of mobility within cytoplasm. NSF DMR08-32802, NIH T32-HL007954, and ONR N000141410538.

  4. Oort spike comets with large perihelion distances (United States)

    Królikowska, Małgorzata; Dybczyński, Piotr A.


    The complete sample of large-perihelion nearly-parabolic comets discovered during the period 1901-2010 is studied, starting with their orbit determination. Next, an orbital evolution that includes three perihelion passages (previous-observed-next) is investigated in which a full model of Galactic perturbations and perturbations from passing stars is incorporated. We show that the distribution of planetary perturbations suffered by actual large-perihelion comets during their passage through the Solar system has a deep, unexpected minimum around zero, which indicates a lack of 'almost unperturbed' comets. Using a series of simulations we show that this deep well is moderately resistant to some diffusion of the orbital elements of the analysed comets. It seems reasonable to assert that the observed stream of these large-perihelion comets experienced a series of specific planetary configurations when passing through the planetary zone. An analysis of the past dynamics of these comets clearly shows that dynamically new comets can appear only when their original semimajor axes are greater than 20 000 au. On the other hand, dynamically old comets are completely absent for semimajor axes longer than 40 000 au. We demonstrate that the observed 1/aori-distribution exhibits a local minimum separating dynamically new from dynamically old comets. Long-term dynamical studies reveal a wide variety of orbital behaviour. Several interesting examples of the action of passing stars are also described, in particular the impact of Gliese 710, which will pass close to the Sun in the future. However, none of the obtained stellar perturbations is sufficient to change the dynamical status of the analysed comets.

  5. High resolution ultrasound and photoacoustic imaging of single cells

    Directory of Open Access Journals (Sweden)

    Eric M. Strohm


    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.

  6. Microfluidic single-cell technology in immunology and antibody screening. (United States)

    Seah, Yu Fen Samantha; Hu, Hongxing; Merten, Christoph A


    Single-cell technology has a major impact on the field of immunology. It enables the kinetics and logic of immune signaling and immune cell migration to be elucidated, facilitates antibody screening and allows massively parallelized analysis of B- and T-cell repertoires. Impressive progress has been made over the last decade, strongly boosted by microfluidic approaches. In this review, we summarize the most powerful microfluidic systems based on continuous flow, nanowells, valves and droplets and we analyze their benefits for phenotypic characterization, drug discovery and next generation sequencing experiments. We describe current limitations and provide an outlook on important future applications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Single-cell technologies in molecular marine studies

    KAUST Repository

    Kodzius, Rimantas


    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.

  8. Single-cell Raman spectroscopy of irradiated tumour cells (United States)

    Matthews, Quinn

    This work describes the development and application of a novel combination of single-cell Raman spectroscopy (RS), automated data processing, and principal component analysis (PCA) for investigating radiation induced biochemical responses in human tumour cells. The developed techniques are first validated for the analysis of large data sets (˜200 spectra) obtained from single cells. The effectiveness and robustness of the automated data processing methods is demonstrated, and potential pitfalls that may arise during the implementation of such methods are identified. The techniques are first applied to investigate the inherent sources of spectral variability between single cells of a human prostate tumour cell line (DU145) cultured in vitro. PCA is used to identify spectral differences that correlate with cell cycle progression and the changing confluency of a cell culture during the first 3-4 days after sub-culturing. Spectral variability arising from cell cycle progression is (i) expressed as varying intensities of protein and nucleic acid features relative to lipid features, (ii) well correlated with known biochemical changes in cells as they progress through the cell cycle, and (iii) shown to be the most significant source of inherent spectral variability between cells. This characterization provides a foundation for interpreting spectral variability in subsequent studies. The techniques are then applied to study the effects of ionizing radiation on human tumour cells. DU145 cells are cultured in vitro and irradiated to doses between 15 and 50 Gy with single fractions of 6 MV photons from a medical linear accelerator. Raman spectra are acquired from irradiated and unirradiated cells, up to 5 days post-irradiation. PCA is used to distinguish radiation induced spectral changes from inherent sources of spectral variability, such as those arising from cell cycle. Radiation induced spectral changes are found to correlate with both the irradiated dose and the

  9. Single-cell mechanics: the parallel plates technique. (United States)

    Bufi, Nathalie; Durand-Smet, Pauline; Asnacios, Atef


    We describe here the parallel plates technique which enables quantifying single-cell mechanics, either passive (cell deformability) or active (whole-cell traction forces). Based on the bending of glass microplates of calibrated stiffness, it is easy to implement on any microscope, and benefits from protocols and equipment already used in biology labs (coating of glass slides, pipette pullers, micromanipulators, etc.). We first present the principle of the technique, the design and calibration of the microplates, and various surface coatings corresponding to different cell-substrate interactions. Then we detail the specific cell preparation for the assays, and the different mechanical assays that can be carried out. Finally, we discuss the possible technical simplifications and the specificities of each mechanical protocol, as well as the possibility of extending the use of the parallel plates to investigate the mechanics of cell aggregates or tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Single-cell analysis of G-protein signal transduction. (United States)

    Clister, Terri; Mehta, Sohum; Zhang, Jin


    The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Single-cell phospho-protein analysis by flow cytometry. (United States)

    Schulz, Kenneth R; Danna, Erika A; Krutzik, Peter O; Nolan, Garry P


    This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings. © 2012 by John Wiley & Sons, Inc.

  12. Production strategies and applications of microbial single cell oils

    Directory of Open Access Journals (Sweden)

    Katrin Ochsenreither


    Full Text Available Polyunsaturated fatty acids (PUFAs of the -3 and -6 class (e.g. -linolenic acid, linoleic acid are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF or solid state fermentation (SSF. The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g. medium, pH-value, temperature, aeration, nitrogen source. From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids

  13. Production Strategies and Applications of Microbial Single Cell Oils. (United States)

    Ochsenreither, Katrin; Glück, Claudia; Stressler, Timo; Fischer, Lutz; Syldatk, Christoph


    Polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 class (e.g., α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogen source). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty

  14. A photoacoustic technique to measure the properties of single cells (United States)

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


    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.

  15. 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.

  16. Elastic hydrogel as a sensor for detection of mechanical stress generated by single cells grown in three-dimensional environment. (United States)

    Huang, Jianyong; Wang, Liangli; Xiong, Chunyang; Yuan, Fan


    Cell volume growth occurs in all living tissues. The growth exerts mechanical stresses on surrounding tissues that may alter tissue microenvironment, and have significant implications in health and diseases. However, the level of growth stress generated by single cells in three-dimensional (3D) environment remains to be determined. To this end, we developed a growth force microscopy technique to determine 3D distribution of the stress. The technique was based on encapsulation of cells in elastic hydrogels, and involved 3D particle tracking and mechanical analysis of gel deformation. Data from the study demonstrated that the growth stress was dynamic, and the stress distribution at the gel-cell interface was correlated inversely to the mean surface curvature or the distance to the geometric center of the cell. The stress averaged over the cell surface increased with increasing gel stiffness, suggesting that cells could alter growth stress in response to stiffness change in microenvironment. These findings suggested that the elastic hydrogel-based microscopy technique had a potential to provide new insights into mechanisms of mechanical interactions between cell and its microenvironment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Effect of chronic low dose natural radiation in human peripheral blood mononuclear cells: Evaluation of DNA damage and repair using the alkaline comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P.R. Vivek, E-mail: [Low Level Radiation Research Laboratory, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, IRE Campus, Beach Road, Kollam 691 001, Kerala (India); Seshadri, M. [Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Jaikrishan, G. [Low Level Radiation Research Laboratory, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, IRE Campus, Beach Road, Kollam 691 001, Kerala (India); Das, Birajalaxmi [Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India)


    Highlights: • Effect of chronic low dose natural radiation in radio adaptive response studied. • PBMCs of subjects from NLNRA and HLNRA were challenged with gamma radiation. • DNA damage and repair in PBMCs was compared using the alkaline comet assay. • Significant reduction in DNA damage in subjects of high dose group from HLNRA noted. • Probable induction of an in vivo radio adaptive response in subjects from HLNRA. - Abstract: This study investigates whether peripheral blood mononuclear cells (PBMCs) from inhabitants of Kerala in southwest India, exposed to chronic low dose natural radiation in vivo (>1 mSv year{sup −1}), respond with a radioadaptive response to a challenging dose of gamma radiation. Toward this goal, PBMCs isolated from 77 subjects from high-level natural radiation areas (HLNRA) and 37 subjects from a nearby normal level natural radiation area (NLNRA) were challenged with 2 Gy and 4 Gy gamma radiation. Subjects from HLNRA were classified based on the mean annual effective dose received, into low dose group (LDG) and high dose group (HDG) with mean annual effective doses of 2.69 mSv (N = 43, range 1.07 mSv year{sup −1} to 5.55 mSv year{sup −1}) and 9.62 mSv (N = 34, range 6.07 mSv year{sup −1} to17.41 mSv year{sup −1}), respectively. DNA strand breaks and repair kinetics (at 7 min, 15 min and 30 min after 4 Gy) were evaluated using the alkaline single cell gel electrophoresis (comet) assay. Initial levels of DNA strand breaks observed after either a 2 Gy or a 4 Gy challenging dose were significantly lower in subjects of the HDG from HLNRA compared to subjects of NLNRA (2 Gy, P = 0.01; 4 Gy, P = 0.02) and LDG (2 Gy P = 0.01; 4 Gy, P = 0.05). Subjects of HDG from HLNRA showed enhanced rejoining of DNA strand breaks (HDG/NLNRA, P = 0.06) during the early stage of repair (within 7 min). However at later times a similar rate of rejoining of strand breaks was observed across the groups (HDG, LDG and NLNRA). Preliminary results from

  18. Comet Hyakutake's close encounter with the Sun (United States)


    LASCO is a joint project between NRL (USA), the Max Planck Institut für Aeronomie (Germany), the Laboratoire d'Astronomie Spatiale (France), and the School of Physics and Space Research at the University of Birmingham (UK). "Such observations require a special instrument in space to suppress the glare of the Sun and reveal the comet and its tails", said Dr. Guenter Brueckner, NRL's principal investigator for LASCO. Scattering of sunlight in the Earth's atmosphere prevented good views from the ground during the comet's "perihelion passage" when it was closest to the Sun. The orbital period of Comet Hyakutake has been estimated to be 10,000 years. Hyakutake is called a "new" comet because it was not seen when, and if, it last visited the solar system. As Hyakutake approaches the Sun, it is being heated enormously. If this is the first visit of the comet, it could be broken into pieces, according to scientists. Images captured by the LASCO instrument have shown that this did not happen when the comet was in LASCO's field of view, which is approximately the size of the constellation Orion. "Comet Hyakutake could have passed through the solar system many times before", said Dr. Brueckner, who is also head of the NRL's Solar Physics Branch. "How many times remains a mystery". Hyakutake's orbit carries it back into the so-called "Oort Cloud" a vast collection of billions of comets that is located 1.4 light years away from the solar system. These comets are presumably the remnants of the cloud from which the solar system were formed billions of years ago. When the comet enters the outer atmosphere of the Sun, it begins to react with the Sun's environment and can be used as a "probe" of the solar corona. LASCO images show the head of the comet, and clearly visible are three separate tails that behave differently as Hyakutake swings around the Sun. These tails are made of different materials ; dust of different sizes, perhaps chunks of ice and atomic particles, each of

  19. On the reflectance of dust in comets (United States)

    Zubko, Evgenij; Videen, Gorden; Shkuratov, Yuriy; Hines, Dean C.


    Reflectance of cometary dust is a key parameter used in the characterization of comets. In the literature, the reflectance of single-scattering cometary dust particles is widely assumed to be the same as that of the cometary nucleus. We discuss this assumption and demonstrate its inconsistency with photometric observations of comets, laboratory optical measurements, and numerical simulation of light scattering from single-scattering dust particles and particulate surfaces composed of the same particles. We estimate the reflectance of cometary dust particles using a comprehensive physical model of polarization measured in comets over wide range of phase angle and at different wavelengths in the visible. The model predicts that the reflectance of dust in comets inversely correlates with their maximum of positive polarization Pmax. We find that even the darkest dust particles appearing in comets with the highest Pmax, reflect considerably more incident solar-radiation energy, up to 200%, compared to what is thought for cometary nuclei. We also find that the reflectance retrieved from polarimetry in the visible appears in good quantitative accordance with previous estimations from infrared observations of comets. Our findings suggest that the dust production of comets is currently overestimated and may require revision.

  20. The Comet Halley Handbook: An Observer's Guide. Second Edition. (United States)

    Yeomans, Donald K.

    This handbook contains information on: (1) the orbit of comet Halley; (2) the expected physical behavior of comet Halley in 1985-1986, considering brightness estimates, coma diameters, and tail lengths; (3) observing conditions for comet Halley in 1985-1986; and (4) observing conditions for the dust tail of comet Halley in 1985-1986. Additional…

  1. Comet Halley Returns. A Teacher's Guide, 1985-1986. (United States)

    Chapman, Robert D.; Bondurant, R. Lynn, Jr.

    This booklet was designed as an aid for elementary and secondary school teachers. It is divided into two distinct parts. Part I is a brief tutorial which introduces some of the most important concepts about comets. Areas addressed include: the historical importance of Comet Halley; how comets are found and names; cometary orbits; what Comet Halley…


    Energy Technology Data Exchange (ETDEWEB)

    Jewitt, David, E-mail: [Department of Earth, Planetary and Space Sciences, UCLA, 595 Charles Young Drive East, Los Angeles, CA 90095-1567 (United States)


    Most comets are volatile-rich bodies that have recently entered the inner solar system following long-term storage in the Kuiper belt and the Oort cloud reservoirs. These reservoirs feed several distinct, short-lived “small body” populations. Here, we present new measurements of the optical colors of cometary and comet-related bodies including long-period (Oort cloud) comets, Damocloids (probable inactive nuclei of long-period comets) and Centaurs (recent escapees from the Kuiper belt and precursors to the Jupiter family comets). We combine the new measurements with published data on short-period comets, Jovian Trojans and Kuiper belt objects to examine the color systematics of the comet-related populations. We find that the mean optical colors of the dust in short-period and long-period comets are identical within the uncertainties of measurement, as are the colors of the dust and of the underlying nuclei. These populations show no evidence for scattering by optically small particles or for compositional gradients, even at the largest distances from the Sun, and no evidence for ultrared matter. Consistent with earlier work, ultrared surfaces are common in the Kuiper belt and on the Centaurs, but not in other small body populations, suggesting that this material is hidden or destroyed upon entry to the inner solar system. The onset of activity in the Centaurs and the disappearance of the ultrared matter in this population begin at about the same perihelion distance (∼10 AU), suggesting that the two are related. Blanketing of primordial surface materials by the fallback of sub-orbital ejecta, for which we calculate a very short timescale, is the likely mechanism. The same process should operate on any mass-losing body, explaining the absence of ultrared surface material in the entire comet population.

  3. A Post-Stardust Mission View of Jupiter Family Comets (United States)

    Zolensky, M.


    Before the Stardust Mission, many persons (including the mission team) believed that comet nuclei would be geologically boring objects. Most believed that comet nucleus mineralogy would be close or identical to the chondritic interplanetary dust particles (IDPs), or perhaps contain mainly amorphous nebular condensates or that comets might even be composed mainly of preserved presolar material [1]. Amazingly, the results for Comet Wild 2 (a Jupiter class comet) were entirely different. Whether this particular comet will ultimately be shown to be typical or atypical will not be known for a rather long time, so we describe our new view of comets from the rather limited perspective of this single mission.

  4. MicroBioRobots for single cell manipulation (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

  5. A Comet Engulfs Mars: MAVEN Observations of Comet Siding Spring's Influence on the Martian Magnetosphere (United States)

    Espley, Jared R.; Dibraccio, Gina A.; Connerney, John E. P.; Brain, David; Gruesbeck, Jacob; Soobiah, Yasir; Halekas, Jasper S.; Combi, Michael; Luhmann, Janet; Ma, Yingjuan


    The nucleus of comet C/2013 A1 (Siding Spring) passed within 141,000?km of Mars on 19 October 2014. Thus, the cometary coma and the plasma it produces washed over Mars for several hours producing significant effects in the Martian magnetosphere and upper atmosphere. We present observations from Mars Atmosphere and Volatile EvolutioN's (MAVEN's) particles and field's instruments that show the Martian magnetosphere was severely distorted during the comet's passage. We note four specific major effects: (1) a variable induced magnetospheric boundary, (2) a strong rotation of the magnetic field as the comet approached, (3) severely distorted and disordered ionospheric magnetic fields during the comet's closest approach, and (4) unusually strong magnetosheath turbulence lasting hours after the comet left. We argue that the comet produced effects comparable to that of a large solar storm (in terms of incident energy) and that our results are therefore important for future studies of atmospheric escape, MAVEN's primary science objective.

  6. Comet 'Bites the Dust' Around Dead Star (United States)


    [figure removed for brevity, see original site] Infrared Spectrometer Graph This artist's concept illustrates a comet being torn to shreds around a dead star, or white dwarf, called G29-38. NASA's Spitzer Space Telescope observed a cloud of dust around this white dwarf that may have been generated from this type of comet disruption. The findings suggest that a host of other comet survivors may still orbit in this long-dead solar system. The white dwarf G29-38 began life as a star that was about three times as massive as our sun. Its death involved the same steps that the sun will ultimately undergo billions of years from now. According to theory, the G29-38 star became brighter and brighter as it aged, until it bloated up into a dying star called a red giant. This red giant was large enough to engulf and evaporate any terrestrial planets like Earth that happened to be in its way. Later, the red giant shed its outer atmosphere, leaving behind a shrunken skeleton of star, called a white dwarf. If the star did host a planetary system, outer planets akin to Jupiter and Neptune and a remote ring of icy comets would remain. The Spitzer observations provide observational evidence for this orbiting outpost of comet survivors. Astronomers speculate that one such comet was knocked into the inner regions of G29-38, possibly by an outer planet. As the comet approached very close to the white dwarf, it may have been torn apart by the star's tidal forces. Eventually, all that would be left of the comet is a disk of dust. This illustration shows a comet in the process of being pulverized: part of it still exists as a chain of small clumps, while the rest has already spread out into a dusty disk. Comet Shoemaker-Levy 9 broke apart in a similar fashion when it plunged into Jupiter in 1994. Evidence for Comets Found in Dead Star's Dust The graph of data, or spectrum, from NASA's Spitzer Space Telescope indicates that a dead star, or white dwarf, called G29-38, is shrouded by a cloud

  7. Chromatin in fractal globule state: evidence from comet assay

    Directory of Open Access Journals (Sweden)

    Afanasieva K. S.


    Full Text Available At higher order levels chromatin is organized into loops that appear as a result of contacts between distant loci. The aim of this work was to investigate the length distribution of the DNA loops in nucleoids obtained after lysis of either whole cells or isolated cell nuclei. Methods. We used single cell gel electrophoresis to analyze the kinetics of the DNA loop migration from the two nucleoid types. Results. The kinetics of the DNA exit was found to have specific features for the two types of nucleoids. At the same time, in both cases, the DNA amount in the electrophoretic track depends linearly on the length of the longest loops in the track. Conclusions. We have concluded that for the loops up to ~ 100 kb the length distribution appears to be consistent with the fractal globule organization.

  8. A New Orbit for Comet C/1865 B1 (Great Southern Comet of 1865) (United States)

    Branham, Richard L., Jr.


    Comet C/1865 B1 (Great southern comet of 1865), observed only in the southern hemisphere, is one of a large number of comets with parabolic orbits. Given that there are 202 observations in right ascension and 165 in declination it proves possible to calculate a better orbit than that Körber published in 1887, the orbit used in various catalogs and data bases. C/1865 B1's orbit is hyperbolic and statistically distinguishable from a parabola. This object, therefore, cannot be considered an NEO. The comet has a small perihelion distance of 0.026 AU.

  9. The role of nanotechnology in single-cell detection: a review. (United States)

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


    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.

  10. 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.


    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

  11. Evaluation of single cell protein for nutrition of farm animals

    Energy Technology Data Exchange (ETDEWEB)

    Oslage, H.J.; Schulz, E.


    For the production of microorganisms with high content of protein technologies on the basis of carbon rich substrates have been developed during the past years. Thus, signification of Single Cell Protein (SCP) for nutrition of farm animals has changed. While, in former times, yeasts were added only in small portions (1-2%) as vitamin supplementation today it is the aim to use microbial biomass as a protein component. The use of SCP as a feedstuff requires a careful physiological and toxicological evaluation as well as extensive investigations of possible use and frontiers of those products for farm animals. Topic of this work were bacteria, bred on methanol as well as yeasts, grown on alcanes and on whey/lactic acid respectively. SCP is preferently used as a feedstuff for poultry, pigs, calves and fishes. Digestibility and utilisation of protein is good till very good, for the a.m. animals, digestibility being between 75-93% and net protein utilisation (NPU) being between 60-76%. In rations of young animals (chicken, piglets and calves) contents of 5-10% SCP have been proved to be without any negative effect on acceptance, body gain, feed utilisation and mortality. For older animals SCP can be used as the only protein source beside the basic feedstuffs.

  12. An RF input coupler for a superconducting single cell cavity

    International Nuclear Information System (INIS)

    Fechner, B.; Ouchi, Nobuo; Kusano, Joichi; Mizumoto, Motoharu; Mukugi, Ken; Krawczyk, F.


    Japan Atomic Energy Research Institute proposes a high intensity proton accelerator for the Neutron Science Project. A superconducting linac is a main option for the high energy part of the accelerator. Design and development work for the superconducting accelerating cavities (resonant frequency of 600 MHz) is in progress. Superconducting cavities have an advantage of very high accelerating efficiency because RF wall loss is very small and much of the RF power fed to the cavity is consumed for the beam acceleration. On the other hand, an RF input coupler for the superconducting cavity has to be matched to the beam loading. Therefore, estimation of coupling coefficient or external quality factor (Qext) of the RF input coupler is important for the design of the couplers. In this work, Qext's were calculated by the electromagnetic analysis code (MAFIA) and were compared with those by the measurements. A β (ratio of the particle velocity to the light velocity) = 0.5 single-cell cavity with either axial coupler or side coupler was used in this work. In the experiments, a model cavity made by copper is applied. Both 2- and 3-dimensional calculations were performed in the axial coupler geometry and the results were compared. The agreements between calculated and measured values are good and this method for calculation of Qext is confirmed to be proper for the design of the RF input couplers. (author)

  13. A microfluidic platform for regulating signal transduction in single cells (United States)

    Wong, Pak Kin; Yu, Fuqu; Sun, Ren; Ho, Chih-Ming


    Recent progress in micro cell culture systems has lead to new approaches in cell biology studies. Using micro devices for cell culturing possesses distinctive advantages over traditional methods. Length scale matching facilitates manipulation and detection at the single cell level. Previously, we have demonstrated generation of various stimulations such as spatial chemical gradient, electric field, and shear stress to study the dynamic responses of individual cells. Dynamic stimulations and continuous monitoring in a microfluidic system can be useful in studying different aspects of cellular process. In this work, we present a microfluidic platform for regulating nuclear factor kappa B (NF-kB) signal transduction in human embryonic kidney 293T cells. Time-varying bio-chemical stimulants, such as interleukin 1 and tumor necrosis factor, are introduced into the microchannel to activate the NF-kB signaling pathway. The dynamic responses of individual cells are monitored with the expression of reporter gene, green fluorescent protein. Regulation of the NF-kB activity is successfully demonstrated. This work is supported by CMISE through NASA URETI program.

  14. 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


    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.

  15. Decoding speech perception from single cell activity in humans. (United States)

    Ossmy, Ori; Fried, Itzhak; Mukamel, Roy


    Deciphering the content of continuous speech is a challenging task performed daily by the human brain. Here, we tested whether activity of single cells in auditory cortex could be used to support such a task. We recorded neural activity from auditory cortex of two neurosurgical patients while presented with a short video segment containing speech. Population spiking activity (~20 cells per patient) allowed detection of word onset and decoding the identity of perceived words with significantly high accuracy levels. Oscillation phase of local field potentials (8-12Hz) also allowed decoding word identity although with lower accuracy levels. Our results provide evidence that the spiking activity of a relatively small population of cells in human primary auditory cortex contains significant information for classification of words in ongoing speech. Given previous evidence for overlapping neural representation during speech perception and production, this may have implications for developing brain-machine interfaces for patients with deficits in speech production. Copyright © 2015 Elsevier Inc. All rights reserved.

  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


    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. Opto-acoustic microscopy reveals adhesion mechanics of single cells (United States)

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


    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.

  18. Single-Cell Phenotype Classification Using Deep Convolutional Neural Networks. (United States)

    Dürr, Oliver; Sick, Beate


    Deep learning methods are currently outperforming traditional state-of-the-art computer vision algorithms in diverse applications and recently even surpassed human performance in object recognition. Here we demonstrate the potential of deep learning methods to high-content screening-based phenotype classification. We trained a deep learning classifier in the form of convolutional neural networks with approximately 40,000 publicly available single-cell images from samples treated with compounds from four classes known to lead to different phenotypes. The input data consisted of multichannel images. The construction of appropriate feature definitions was part of the training and carried out by the convolutional network, without the need for expert knowledge or handcrafted features. We compare our results against the recent state-of-the-art pipeline in which predefined features are extracted from each cell using specialized software and then fed into various machine learning algorithms (support vector machine, Fisher linear discriminant, random forest) for classification. The performance of all classification approaches is evaluated on an untouched test image set with known phenotype classes. Compared to the best reference machine learning algorithm, the misclassification rate is reduced from 8.9% to 6.6%. © 2016 Society for Laboratory Automation and Screening.

  19. Single cell detection using a magnetic zigzag nanowire biosensor. (United States)

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


    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.

  20. Using single cell sequencing data to model the evolutionary history of a tumor


    Kim, Kyung In; Simon, Richard


    Background The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumo...

  1. Reconstructing Cell Lineages from Single-Cell Gene Expression Data: A Pilot Study (United States)


    Reconstructing cell lineages from single -cell gene expression data: a pilot study The goal of this pilot study is to develop novel mathematical...methods, by leveraging tools developed in the bifurcation theory, to infer the underlying cell-state dynamics from single -cell gene expression data. Our...from single -cell gene expression data. The views, opinions and/or findings contained in this report are those of the author(s) and should not contrued

  2. A Comet Surface Sample Return System Project (United States)

    National Aeronautics and Space Administration — The proposed Phase I investigation will focus on the development of spacecraft systems required to obtain a sample from the nucleus of a comet, hermetically seal the...

  3. Laboratory Study on Disconnection Events in Comets (United States)

    Li, Yan-Fei; Li, Yu-Tong; Wang, Wei-Min; Yuan, Da-Wei; et al.


    When comets interacting with solar wind, straight and narrow plasma tails will be often formed. The most remarkable phenomenon of the plasma tails is the disconnection event, in which a plasma tail is uprooted from the comet's head and moves away from the comet. In this paper, the interaction process between a comet and solar wind is simulated by using a laser-driven plasma cloud to hit a cylinder obstacle. A disconnected plasma tail is observed behind the obstacle by optical shadowgraphy and interferometry. Our particle-in-cell simulations show that the diference in thermal velocity between ions and electrons induces an electrostatic field behind the obstacle. This field can lead to the convergence of ions to the central region, resulting in a disconnected plasma tail. This electrostatic field-induced model may be a possible explanation for the disconnection events of cometary tails.

  4. Desenvolvimento e validação de técnica quantitativa de análise de imagem para avaliação do teste do cometa corado pela prata Development and validation of a quantitative image analysis method to evaluate comet assay (silver staining

    Directory of Open Access Journals (Sweden)

    Gabrielli Brianezi


    Full Text Available INTRODUÇÃO: O ensaio do cometa ou técnica da eletroforese de células isoladas é largamente empregado para avaliação de danos e reparo do DNA em células individuais. O material pode ser corado por técnicas de fluorescência ou por sal de prata. Este último apresenta vantagens técnicas, como o tipo de microscópio utilizado e a possibilidade de armazenamento das lâminas. A análise dos cometas pode ser feita de modo visual, porém há a desvantagem da subjetividade dos resultados, que pode ser minimizada por análise digital automatizada. OBJETIVOS: Desenvolvimento e validação de método de análise digital de cometas corados por sal de prata. MÉTODOS: Cinquenta cometas foram fotografados de maneira padronizada e impressos em papel. Além de medidas manualmente, essas imagens foram classificadas em cinco categorias por três avaliadores, antes e depois de pré-processadas automaticamente pelo software ImageJ 1.38x. As estimativas geradas pelos avaliadores foram comparadas quanto sua correlação e reprodutibilidade. Em seguida, foram desenvolvidos algoritmos de análise digital das medidas, com base em filtros estatísticos de mediana e de mínimo. Os valores obtidos foram comparados com os estimados manual e visualmente após o pré-processamento. RESULTADOS: As medidas manuais das imagens pré-processadas apresentaram maior correlação intraclasse do que as imagens preliminares. Os parâmetros automatizados apresentaram alta correlação com as medidas manuais pré-processadas, sugerindo que este sistema aumenta a objetividade da análise, podendo ser utilizado na estimativa dos parâmetros dos cometas. CONCLUSÃO: A presente análise digital proposta para o teste do cometa corado pela prata mostrou-se factível e de melhor reprodutibilidade que a análise visual.BACKGROUND: Comet assay or single cell gel electrophoresis is a useful and widely applied technique for the assessment of DNA damage and repair in individual cells

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

    Directory of Open Access Journals (Sweden)

    Zixi Chen


    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.

  6. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Dovichi, Norman J


    .... Capillary sieving electrophoresis and capillary micellar electrophoresis were used to characterize proteins in single cells in one-dimensional separations, while the two techniques were combined...

  7. Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation) (United States)

    Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi


    Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

  8. Radio Observations of Organics in Comets (United States)

    Milam, Stefanie N.; Charnley, Steven B.; Kuan, Yi-Jehng; Chuang, Yo-Ling; Villanueva, Geronimo; Coulson, Iain; Remijan, Anthony J.


    A major observational challenge in cometary science is to quantify the extent to which chemical compounds can be linked to either interstellar or nebular chemistry. Recently, there have been complimentary observations from multiple facilities to try to unravel the chemical complexity of comets and their origins. Incorporating results from various techniques can gain further insight into the abundances, production rates, distributions, and formation mechanisms of molecules in these objects [I]. Such studies have provided great detail towards molecules with a-typical chemistries, such as H2CO [2]. We report multiwavelength spectral observations of comets from two dynamical families including the JFC 103P/Hartley 2 and a long period comet C/2009 PI (Garradd) with the Arizona Radio Observatory's SMT and 12-m telescopes, as well as the NRAO Greenbank telescope, and the James Clerk Maxwell Telescope. Multiple parent volatiles (e.g. HCN, CH30H, CO) as well as daughter products (e.g, CS and 01-1) have been detected in these objects. We will present a comparison of molecular abundances in these comets to those observed in others, supporting a long-term effort of building a comet taxonomy based on composition. Previous work has revealed a range of abundances of parent species (from "organics-poor" to "organics-rich") with respect to water among comets [3,4,5], however the statistics are not well constrained.

  9. The Comet Radar Explorer Mission (United States)

    Asphaug, Erik; Belton, Mike; Bockelee-Morvan, Dominique; Chesley, Steve; Delbo, Marco; Farnham, Tony; Gim, Yonggyu; Grimm, Robert; Herique, Alain; Kofman, Wlodek; Oberst, Juergen; Orosei, Roberto; Piqueux, Sylvain; Plaut, Jeff; Robinson, Mark; Sava, Paul; Heggy, Essam; Kurth, William; Scheeres, Dan; Denevi, Brett; Turtle, Elizabeth; Weissman, Paul


    Missions to cometary nuclei have revealed major geological surprises: (1) Global scale layers - do these persist through to the interior? Are they a record of primary accretion? (2) Smooth regions - are they landslides originating on the surface? Are they cryovolcanic? (3) Pits - are they impact craters or sublimation pits, or rooted in the interior? Unambiguous answers to these and other questions can be obtained by high definition 3D radar reflection imaging (RRI) of internal structure. RRI can answer many of the great unknowns in planetary science: How do primitive bodies accrete? Are cometary nuclei mostly ice? What drives their spectacular activity and evolution? The Comet Radar Explorer (CORE) mission will image the detailed internal structure of the nucleus of 10P/Tempel 2. This ~16 x 8 x 7 km Jupiter Family Comet (JFC), or its parent body, originated in the outer planets region possibly millions of years before planet formation. CORE arrives post-perihelion and observes the comet’s waning activity from safe distance. Once the nucleus is largely dormant, the spacecraft enters a ~20-km dedicated Radar Mapping Orbit (RMO). The exacting design of the RRI experiment and the precise navigation of RMO will achieve a highly focused 3D radar reflection image of internal structure, to tens of meters resolution, and tomographic images of velocity and attenuation to hundreds of meters resolution, tied to the gravity model and shape. Visible imagers will produce maps of the surface morphology, albedo, color, texture, and photometric response, and images for navigation and shape determination. The cameras will also monitor the structure and dynamics of the coma, and its dusty jets, allowing their correlation in 3D with deep interior structures and surface features. Repeated global high-resolution thermal images will probe the near-surface layers heated by the Sun. Derived maps of thermal inertia will be correlated with the radar boundary response, and photometry and

  10. Modeling bacterial population growth from stochastic single-cell dynamics. (United States)

    Alonso, Antonio A; Molina, Ignacio; Theodoropoulos, Constantinos


    A few bacterial cells may be sufficient to produce a food-borne illness outbreak, provided that they are capable of adapting and proliferating on a food matrix. This is why any quantitative health risk assessment policy must incorporate methods to accurately predict the growth of bacterial populations from a small number of pathogens. In this aim, mathematical models have become a powerful tool. Unfortunately, at low cell concentrations, standard deterministic models fail to predict the fate of the population, essentially because the heterogeneity between individuals becomes relevant. In this work, a stochastic differential equation (SDE) model is proposed to describe variability within single-cell growth and division and to simulate population growth from a given initial number of individuals. We provide evidence of the model ability to explain the observed distributions of times to division, including the lag time produced by the adaptation to the environment, by comparing model predictions with experiments from the literature for Escherichia coli, Listeria innocua, and Salmonella enterica. The model is shown to accurately predict experimental growth population dynamics for both small and large microbial populations. The use of stochastic models for the estimation of parameters to successfully fit experimental data is a particularly challenging problem. For instance, if Monte Carlo methods are employed to model the required distributions of times to division, the parameter estimation problem can become numerically intractable. We overcame this limitation by converting the stochastic description to a partial differential equation (backward Kolmogorov) instead, which relates to the distribution of division times. Contrary to previous stochastic formulations based on random parameters, the present model is capable of explaining the variability observed in populations that result from the growth of a small number of initial cells as well as the lack of it compared to

  11. Flagellates as model system for gravity detection of single cells (United States)

    Lebert, Michael; Richter, Peter; Daiker, Viktor; Schuster, Martin; Tebart, Jenny; Strauch, Sebastian M.; Donat-Peter, H.

    Euglena gracilis is a unicellular, photosynthetic organism which uses light and gravity as en-vironmental hints to reach and stay in horizons of the water column which are optimal for growth and reproduction. The orientation in respect to light (so called positive and nega-tive phototaxis, i.e. movement toward or away of a light source) was well known and fairly good understood. In contrast, knowledge about the movement away from the centre of gravity (negative gravitaxis) was rather scarce. Over a century it was unclear whether orientation in respect to the gravity vector is based on a physical or a physiological mechanism. Recent results clearly favour the latter. Knock-down mutants (RNAi) were characterized which define certain key components of the gravitactic signal transduction chain. These key components include a TRP-like channel, a gravitaxis-specific calmodulin and a protein kinase A. The molecular characterization of these components is currently performed and will be presented. Euglena is not only a model system for the close understanding of gravity detection in single cells, but can also be used as photosynthetic component, i.e. oxygen source and carbon dioxide as well as nitrogenic components sink in Closed Environmental Systems (CES). Due CES are systems of choice in times of scarce flight opportunities. They allow a massive sample sharing and combine possibilities to do microgravity research for biologists but also for engineers, physicists and material scientists. Recent attempts include Aquacells and Omegahab. In the near future miniaturized systems (Chinese ShenZhou) as well as advanced CES will be flown or tested, respectively. Current attempts and plans will be presented.

  12. Transport and Capture of Comets (United States)

    Ross, S. D.; Koon, W. S.; Lo, M. W.; Marsden, J. E.


    The dynamics of comets and other solar system objects which have a three-body energy close to that of the collinear libration points are known to exhibit a complicated array of behaviors such as rapid transition between the interior and exterior Hill's regions, temporary capture, and collision. The invariant manifold structures of the collinear libration points for the restricted three-body problem, which exist for a range of energies, provide the framework for understanding these transport phenomena from a geometric point of view. In particular, the stable and unstable invariant manifold "tubes" associated to libration point orbits are the phase space conduits transporting material to and from the smaller primary body (e.g., Jupiter), and between primary bodies for separate three-body systems (e.g., Saturn and Jupiter). This point of view has worked well in describing the planar circular restricted three-body problem. The current work seeks to extend the results to three degrees of freedom. This work was supported by the National Science Foundation Grant No. KDI/ATM-9873133 under a contract with the Jet Propulsion Laboratory, NASA.

  13. Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

    Directory of Open Access Journals (Sweden)

    Jan Mueller


    Full Text Available Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

  14. Reasonable threshold value used to segment the individual comet from the comet assay image

    International Nuclear Information System (INIS)

    Yan Xuekun; Chen Ying; Du Jie; Zhang Xueqing; Luo Yisheng


    Reasonable segmentation of the individual comet contour from the Comet Assay (CA) images is the precondition for all of parameters analysis during the automatic analyzing for the CA. The Otsu method and several arithmetic operators for image segmentation, such as Sobel, Prewitt, Roberts and Canny were used to segment the comet contour, and characters of the CA images were analyzed firstly. And then the segmentation methods which had been adopted in the software for CA automatic analysis, such as the CASP, the TriTek CometScore TM , were put for-ward and compared. At last, a two-step procedure for threshold calculation based on image-content analysis is adopted to segment the individual comet from the CA images, and several principles for the segmentation are put forward too.(authors)

  15. 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


    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

  16. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity. (United States)

    Clark, Stephen J; Lee, Heather J; Smallwood, Sébastien A; Kelsey, Gavin; Reik, Wolf


    Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

  17. 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

  18. Comet Tempel 1 Went Back to Sleep (United States)


    Astronomers Having Used ESO Telescopes Start Analysing Unique Dataset on the Comet Following the Deep Impact Mission Ten days after part of the Deep Impact spacecraft plunged onto Comet Tempel 1 with the aim to create a crater and expose pristine material from beneath the surface, astronomers are back in the ESO Offices in Santiago, after more than a week of observing at the ESO La Silla Paranal Observatory. In this unprecedented observing campaign - among the most ambitious ever conducted by a single observatory - the astronomers have collected a large amount of invaluable data on this comet. The astronomers have now started the lengthy process of data reduction and analysis. Being all together in a single place, and in close contacts with the space mission' scientific team, they will try to assemble a clear picture of the comet and of the impact. The ESO observations were part of a worldwide campaign to observe this unique experiment. During the campaign, ESO was connected by phone, email, and videoconference with colleagues in all major observatories worldwide, and data were freely exchanged between the different groups. This unique collaborative spirit provides astronomers with data taken almost around the clock during several days and this, with the largest variety of instruments, making the Deep Impact observing campaign one of the most successful of its kind, and thereby, ensuring the greatest scientific outcome. From the current analysis, it appears most likely that the impactor did not create a large new zone of activity and may have failed to liberate a large quantity of pristine material from beneath the surface. ESO PR Photo 22/05 ESO PR Photo 22/05 Evolution of Comet Tempel 1 (FORS2/VLT) [Preview - JPEG: 400 x 701 pix - 128k] [Normal - JPEG: 800 x 1401 pix - 357k] ESO PR Photo 22/05 Animated Gif Caption: ESO PR Photo 22/05 shows the evolution of Comet Tempel 1 as observed with the FORS2 instrument on Antu (VLT). The images obtained at the VLT show that

  19. 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...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live....../Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where...

  20. Unravelling biology and shifting paradigms in cancer with single-cell sequencing. (United States)

    Baslan, Timour; Hicks, James


    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.

  1. A GREAT search for Deuterium in Comets (United States)

    Mumma, Michael


    Comets are understood to be the most pristine bodies in the Solar System. Their compositions reflect the chemical state of materials at the very earliest evolutionary stages of the protosolar nebula and, as such, they provide detailed insight into the physical and chemical processes operating in planet-forming disks. Isotopic fractionation ratios of the molecular ices in the nucleus are regarded as signatures of formation processes. These ratios provide unique information on the natal heritage of those ices, and can also test the proposal that Earth's water and other volatiles were delivered by cometary bombardment. Measurement of deuterium fractionation ratios is thus a major goal in contemporary cometary science and the D/H ratio of water - the dominant volatile in comets - holds great promise for testing the formation history of cometary matter. The D/H ratio in cometary water has been measured in only eight comets. Seven were from the Oort Cloud reservoir and the D/H ratio was about twice that of the Earth's oceans. However, the recent Herschel measurement of HDO/H2O in 103P/Hartley-2 (the first from the Kuiper Belt) was consistent with exogenous delivery of Earth's water by comets. Outstanding questions remain: are cometary HDO/H2O ratios consistent with current theories of nebular chemical evolution or with an interstellar origin? Does the HDO/H2O ratio vary substantially among comet populations? Hartley-2 is the only Kuiper Belt comet with measured HDO/H2O, are there comets with similar ratios in the Oort cloud? These questions can only be addressed by measuring HDO/H2O ratios in many more suitable bright comets. We therefore propose to measure the D/H ratio in water in a suitable target-of-opportunity comet by performing observations of HDO and OH with the GREAT spectrometer on SOFIA. A multi-wavelength, ground-based observing campaign will also be conducted in support of the airborne observations.

  2. Abundant Solar Nebula Solids in Comets (United States)

    Messenger, S.; Keller, L. P.; Nakamura-Messenger, K.; Nguyen, A. N.; Clemett, S.


    Comets have been proposed to consist of unprocessed interstellar materials together with a variable amount of thermally annealed interstellar grains. Recent studies of cometary solids in the laboratory have shown that comets instead consist of a wide range of materials from across the protoplanetary disk, in addition to a minor complement of interstellar materials. These advances were made possible by the return of direct samples of comet 81P/Wild 2 coma dust by the NASA Stardust mission and recent advances in microscale analytical techniques. Isotopic studies of 'cometary' chondritic porous interplanetary dust particles (CP-IDPs) and comet 81P/Wild 2 Stardust samples show that preserved interstellar materials are more abundant in comets than in any class of meteorite. Identified interstellar materials include sub-micron-sized presolar silicates, oxides, and SiC dust grains and some fraction of the organic material that binds the samples together. Presolar grain abundances reach 1 weight percentage in the most stardust-rich CP-IDPs, 50 times greater than in meteorites. Yet, order of magnitude variations in presolar grain abundances among CP-IDPs suggest cometary solids experienced significant variations in the degree of processing in the solar nebula. Comets contain a surprisingly high abundance of nebular solids formed or altered at high temperatures. Comet 81P/Wild 2 samples include 10-40 micron-sized, refractory Ca- Al-rich inclusion (CAI)-, chondrule-, and ameboid olivine aggregate (AOA)-like materials. The O isotopic compositions of these refractory materials are remarkably similar to their meteoritic counterparts, ranging from 5 percent enrichments in (sup 16) O to near-terrestrial values. Comet 81P/Wild 2 and CP-IDPs also contain abundant Mg-Fe crystalline and amorphous silicates whose O isotopic compositions are also consistent with Solar System origins. Unlike meteorites, that are dominated by locally-produced materials, comets appear to be composed of

  3. Migration of Interplanetary Dust and Comets (United States)

    Ipatov, S. I.; Mather, J. C.

    Our studies of migration of interplanetary dust and comets were based on the results of integration of the orbital evolution of 15,000 dust particles and 30,000 Jupiter-family comets (JFCs) [1-3]. For asteroidal and cometary particles, the values of the ratio β between the radiation pressure force and the gravitational force varied from 1000 and 1 microns. The probability of a collision of a dust particle started from an asteroid or JFC with the Earth during a lifetime of the particle was maximum at diameter d ˜100 microns. For particles started from asteroids and comet 10P, this maximum probability was ˜0.01. Different studies of migration of dust particles and small bodies testify that the fraction of cometary dust particles of the overall dust population inside Saturn's orbit is considerable and can be dominant: (1) Cometary dust particles produced both inside and outside Jupiter's orbit are needed to explain the observed constant number density of dust particles at 3-18 AU. The number density of migrating trans-Neptunian particles near Jupiter's orbit is smaller by a factor of several than that beyond Saturn's orbit. Only a small fraction of asteroidal particles can get outside Jupiter's orbit. (2) Some (less than 0.1%) JFCs can reach typical near-Earth object orbits and remain there for millions of years. Dynamical lifetimes of most of the former JFCs that have typical near-Earth object orbits are about 106 -109 yr, so during most of these times they were extinct comets. Such former comets could disintegrate and produce a lot of mini-comets and dust. (3) Comparison of the velocities of zodiacal dust particles (velocities of MgI line) based on the distributions of particles over their orbital elements obtained in our runs [3-4] with the velocities obtained at the WHAM observations shows that only asteroidal dust particles cannot explain these observations, and particles produced by comets, including high-eccentricity comets, are needed for such explanation

  4. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell. (United States)

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


    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.

  5. Removing water from gels

    International Nuclear Information System (INIS)

    Lane, E.S.; Winter, J.A.


    Water is removed from a gel material by contacting the gel material with an organic liquid and contacting the organic liquid with a gas such that water is taken up by the gas. The invention, in one embodiment, may be used to dry gel materials whilst maintaining an open porous network therein. In one example, the invention is applied to gel precipitated spheres containing uranium and plutonium. (author)

  6. Polymer gels and networks

    National Research Council Canada - National Science Library

    Osada, Yoshihito; Khokhlov, A. R


    ... or magnetic field, etc.). It was realized that not only can polymer gels absorb and hold a considerable volume of liquids, but they can also be forced to expel the absorbed liquid in a controlled manner. Of particular interest are hydrogels, i.e., polymer gels, which swell extensively in water. The most common hydrogels are polyelectrolyte gels: ...

  7. Localization of specific sequences and DNA single-strand breaks in individual UV-A-irradiated human lymphocytes by COMET FISH (United States)

    Bock, Claudia; Rapp, Alexander; Dittmar, Heike; Monajembashi, Shamci; Greulich, Karl-Otto


    The COMET assay, a single cell electrophoresis technique which allows to separate electrophoretically fractionated DNA according to size has been combined with fluorescence in situ hybridization (FISH) which allows to localize specific genes or gene regions. This combination (COMET FISH) allows the detection of DNA single strand breaks in specific regions of the genome of human lymphocytes at the single cell level. Various types of DNA probes, e.g. centromere-, (alpha) - satellite-, telomere-, whole chromosome-, single copy- and region specific DNA probes have been used to investigate whether the UV-A induced DNA single strand breaks are distributed randomly all over the human genome or induced at specific sites ('hot spots'). In the investigated human peripheral blood lymphocytes all but one centromere reveal low sensitivity for UV-A irradiation (500 kJ/m2), while telomeres are randomly distributed over COMET heads and tails. The human chromosome 1 is fractionated by irradiation, but remains in the COMET head, indicating an only moderate degree of fractionation. Among three tested single copy probes, c- myc, p53 and p58, the p53 gene located on chromosome 17p13.1 and the p58 gene (1p36) appear to be located in UV-A stable regions of the human genome in 95% of 65 investigated lymphocytes. In contrast, the c-myc proto-oncogene (8q24) is found in the COMET tail in 90% of the 27 investigated lymphocytes and thus appears to be more sensitive to UV-A irradiation.


    National Aeronautics and Space Administration — The McDonald Observatory Faint Comet Survey data set presents spectral data from 152 observations of 17 comets taken using the Intensified Dissector Scanner...


    National Aeronautics and Space Administration — This is a dataset that compiles the data from various papers, which reported observations of brightness changes in comets that allowed to produce the comet...

  10. Dynamical evolution and disintegration of comets (United States)

    Kresak, L.

    Current concepts of the origin and evolution of comets are reviewed. The place of their formation from which they have been delivered into the Oort reservoir is still an open problem, but the region of the outermost planets appears most probable. The interplay of stellar and planetary perturbations can be traced by model computations which reveal both the general trends and the variety of individual evolutionary paths. The present structure of the system of comets is controlled by the dynamical evolution of its individual members, limited by their physical aging by disintegration. Where the lifetimes are short, as in the Jupiter family of short-period comets, an equilibrium between elimination and replenishment is established. The role of different destructive processes and the resulting survival times are discussed.

  11. Comets As Objects of High Energy Astrophysics (United States)

    Ibadov, S.


    Strong soft X-ray emission from comet Hyakutake C/1996 B2 was discovered with ROSAT in March 27, 1996 (Lisse et al. 1996, Science 274, 205-209) and the results of a theoretical approach (Ibadov 1990, Icarus 86, 283-288) served as a motive for that observations (Dennerl, Lisse and Truemper 1998, Private Communications). It is now well established that comets emit EUV and X-rays regularly (Dennerl, Englhauser and Truemper 1997, Science 277, 1625-1630; Dennerl 1998, Proc. 16th Int. Conf. Atomic Physics, Windsor, Ontario, Canada). To explain this phenomenon different theoretical models were proposed (Krasnopolsky 1997, Icarus 128, 365-385; Ibadov 1998, Proc. First XMM Workshop, Noordwijk, The Netherlands, and references therein). In the paper the problem of identifying X-ray generation mechanisms in comets will be considered.

  12. The mass disruption of Jupiter Family comets (United States)

    Belton, Michael J. S.


    I show that the size-distribution of small scattered-disk trans-neptunian objects when derived from the observed size-distribution of Jupiter Family comets (JFCs) and other observational constraints implies that a large percentage (94-97%) of newly arrived active comets within a range of 0.2-15.4 km effective radius must physically disrupt, i.e., macroscopically disintegrate, within their median dynamical lifetime. Additional observational constraints include the numbers of dormant and active nuclei in the near-Earth object (NEO) population and the slope of their size distributions. I show that the cumulative power-law slope (-2.86 to -3.15) of the scattered-disk TNO hot population between 0.2 and 15.4 km effective radius is only weakly dependent on the size-dependence of the otherwise unknown disruption mechanism. Evidently, as JFC nuclei from the scattered disk evolve into the inner Solar System only a fraction achieve dormancy while the vast majority of small nuclei (e.g., primarily those with effective radius <2 km) break-up. The percentage disruption rate appears to be comparable with that of the dynamically distinct Oort cloud and Halley type comets (Levison, H.F., Morbidelli, A., Dones, L., Jedicke, R., Wiegert, P.A., Bottke Jr., W.F. [2002]. Science 296, 2212-2215) suggesting that all types of comet nuclei may have similar structural characteristics even though they may have different source regions and thermal histories. The typical disruption rate for a 1 km radius active nucleus is ∼5 × 10-5 disruptions/year and the dormancy rate is typically 3 times less. We also estimate that average fragmentation rates range from 0.01 to 0.04 events/year/comet, somewhat above the lower limit of 0.01 events/year/comet observed by Chen and Jewitt (Chen, J., Jewitt, D.C. [1994]. Icarus 108, 265-271).

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

    Directory of Open Access Journals (Sweden)

    Lihui Ren


    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.

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

    Directory of Open Access Journals (Sweden)

    Naveen Ramalingam


    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.

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

    Directory of Open Access Journals (Sweden)

    Amelia Ahmad Khalili


    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.

  16. 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


    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.

  17. High-throughput microfluidic single-cell digital polymerase chain reaction. (United States)

    White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L


    Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

  18. SINCERITIES: Inferring gene regulatory networks from time-stamped single cell transcriptional expression profiles. (United States)

    Papili Gao, Nan; Ud-Dean, S M Minhaz; Gandrillon, Olivier; Gunawan, Rudiyanto


    Single cell transcriptional profiling opens up a new avenue in studying the functional role of cell-to-cell variability in physiological processes. The analysis of single cell expression profiles creates new challenges due to the distributive nature of the data and the stochastic dynamics of gene transcription process. The reconstruction of gene regulatory networks (GRNs) using single cell transcriptional profiles is particularly challenging, especially when directed gene-gene relationships are desired. We developed SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS) for the inference of GRNs from single cell transcriptional profiles. We focused on time-stamped cross-sectional expression data, commonly generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers directed regulatory relationships among genes by employing regularized linear regression (ridge regression), using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using in silico time-stamped single cell expression data and single cell transcriptional profiles of THP-1 monocytic human leukemia cells. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Moreover, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality. Finally, an application of SINCERITIES to single cell expression data of T2EC chicken erythrocytes pointed to BATF as a candidate novel regulator of erythroid development. The MATLAB and R version of SINCERITIES is freely available from the following websites: and

  19. The use of 1986 Comet Halley observations for space projects (United States)

    Kolyuka, Y. F.; Kudryavtsev, S. M.; Tarasov, V. P.; Tikhonov, V. F.


    The used observations of comet Halley in the current appearance for the improvement of the comet's orbit for purposes of the projects "Vega" and "Pathfinder" are described. The data on comet observations from different observatories and spacecraft "Vega" are analyzed. The improved orbit parameters are given.

  20. To Catch A Comet...Learning From Halley's. (United States)

    National Aeronautics and Space Administration, Washington, DC.

    Comet chronicles and stories extend back over thousands of years. A common theme has been that comets are a major cause of catastrophe and tragedy here on earth. In addition, both Aristotle and Ptolemy believed that comets were phenomena within the earth's atmosphere, and it wasn't until the 16th century, when Danish astronomer Tycho Brache…

  1. 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)


    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.

  2. ZIFA: Dimensionality reduction for zero-inflated single-cell gene expression analysis. (United States)

    Pierson, Emma; Yau, Christopher


    Single-cell RNA-seq data allows insight into normal cellular function and various disease states through molecular characterization of gene expression on the single cell level. Dimensionality reduction of such high-dimensional data sets is essential for visualization and analysis, but single-cell RNA-seq data are challenging for classical dimensionality-reduction methods because of the prevalence of dropout events, which lead to zero-inflated data. Here, we develop a dimensionality-reduction method, (Z)ero (I)nflated (F)actor (A)nalysis (ZIFA), which explicitly models the dropout characteristics, and show that it improves modeling accuracy on simulated and biological data sets.

  3. New insights into human primordial germ cells and early embryonic development from single-cell analysis. (United States)

    Otte, Jörg; Wruck, Wasco; Adjaye, James


    Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications. © 2017 Federation of European Biochemical Societies.

  4. CometQ: An automated tool for the detection and quantification of DNA damage using comet assay image analysis. (United States)

    Ganapathy, Sreelatha; Muraleedharan, Aparna; Sathidevi, Puthumangalathu Savithri; Chand, Parkash; Rajkumar, Ravi Philip


    DNA damage analysis plays an important role in determining the approaches for treatment and prevention of various diseases like cancer, schizophrenia and other heritable diseases. Comet assay is a sensitive and versatile method for DNA damage analysis. The main objective of this work is to implement a fully automated tool for the detection and quantification of DNA damage by analysing comet assay images. The comet assay image analysis consists of four stages: (1) classifier (2) comet segmentation (3) comet partitioning and (4) comet quantification. Main features of the proposed software are the design and development of four comet segmentation methods, and the automatic routing of the input comet assay image to the most suitable one among these methods depending on the type of the image (silver stained or fluorescent stained) as well as the level of DNA damage (heavily damaged or lightly/moderately damaged). A classifier stage, based on support vector machine (SVM) is designed and implemented at the front end, to categorise the input image into one of the above four groups to ensure proper routing. Comet segmentation is followed by comet partitioning which is implemented using a novel technique coined as modified fuzzy clustering. Comet parameters are calculated in the comet quantification stage and are saved in an excel file. Our dataset consists of 600 silver stained images obtained from 40 Schizophrenia patients with different levels of severity, admitted to a tertiary hospital in South India and 56 fluorescent stained images obtained from different internet sources. The performance of "CometQ", the proposed standalone application for automated analysis of comet assay images, is evaluated by a clinical expert and is also compared with that of a most recent and related software-OpenComet. CometQ gave 90.26% positive predictive value (PPV) and 93.34% sensitivity which are much higher than those of OpenComet, especially in the case of silver stained images. The

  5. Transport Phenomena in Gel

    Directory of Open Access Journals (Sweden)

    Masayuki Tokita


    Full Text Available Gel becomes an important class of soft materials since it can be seen in a wide variety of the chemical and the biological systems. The unique properties of gel arise from the structure, namely, the three-dimensional polymer network that is swollen by a huge amount of solvent. Despite the small volume fraction of the polymer network, which is usually only a few percent or less, gel shows the typical properties that belong to solids such as the elasticity. Gel is, therefore, regarded as a dilute solid because its elasticity is much smaller than that of typical solids. Because of the diluted structure, small molecules can pass along the open space of the polymer network. In addition to the viscous resistance of gel fluid, however, the substance experiences resistance due to the polymer network of gel during the transport process. It is, therefore, of importance to study the diffusion of the small molecules in gel as well as the flow of gel fluid itself through the polymer network of gel. It may be natural to assume that the effects of the resistance due to the polymer network of gel depends strongly on the network structure. Therefore, detailed study on the transport processes in and through gel may open a new insight into the relationship between the structure and the transport properties of gel. The two typical transport processes in and through gel, that is, the diffusion of small molecules due to the thermal fluctuations and the flow of gel fluid that is caused by the mechanical pressure gradient will be reviewed.

  6. Every cell is special: genome-wide studies add a new dimension to single-cell biology. (United States)

    Junker, Jan Philipp; van Oudenaarden, Alexander


    Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide single-cell studies. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Every cell is special : genome-wide studies add a new dimension to single-cell biology

    NARCIS (Netherlands)

    Junker, Jan Philipp; van Oudenaarden, Alexander


    Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide single-cell studies.

  8. Sol-Gel Glasses (United States)

    Mukherjee, S. P.


    Multicomponent homogeneous, ultrapure noncrystalline gels/gel derived glasses are promising batch materials for the containerless glass melting experiments in microgravity. Hence, ultrapure, homogeneous gel precursors could be used to: (1) investigate the effect of the container induced nucleation on the glass forming ability of marginally glass forming compositions; and (2) investigate the influence of gravity on the phase separation and coarsening behavior of gel derived glasses in the liquid-liquid immiscibility zone of the nonsilicate systems having a high density phase. The structure and crystallization behavior of gels in the SiO2-GeO2 as a function of gel chemistry and thermal treatment were investigated. As are the chemical principles involved in the distribution of a second network former in silica gel matrix being investigated. The procedures for synthesizing noncrystalline gels/gel-monoliths in the SiO2-GeO2, GeO2-PbO systems were developed. Preliminary investigations on the levitation and thermal treatment of germania silicate gel-monoliths in the Pressure Facility Acoustic Levitator were done.


    Energy Technology Data Exchange (ETDEWEB)

    Haipeng Wang, Guangfeng Cheng, Gianluigi Ciovati, Peter Kneisel, Robert Rimmer, Kai Tian, Larry Turlington, Alireza Nassiri, Geoff Waldschmidt


    After the design optimization of a squashed elliptical shape, single-cell, superconducting (SC) deflecting cavity at 2.815 GHz, a copper prototype has been bench measured to determine its rf properties and the effectiveness of waveguide damping of parasitic modes [1]. RF cold tests were also performed at 2K on niobium single-cell and two-cell prototype cavities. Details of impedance calculation using wakefiled analysis of the single-cell cavity are shown to meet the strict 200 mA beam stability requirement of the Advanced Photon Source (APS) at Argonne National Lab where a total of 16 single-cell cavities will be divided into two cryomodule. The design of higher-order mode (HOM) waveguide damping, the simulations of the Lorenz force detuning, and the prototype of on-cell damping are presented.

  10. Essentials of single-cell analysis concepts, applications and future prospects

    CERN Document Server

    Santra, Tuhin


    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...

  11. Single cell analysis: the new frontier in 'Omics'

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Bodovitz, Steven


    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.

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

  13. 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


    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...

  14. Spectral analysis of the Chandra comet survey

    NARCIS (Netherlands)

    Bodewits, D.; Christian, D. J.; Torney, M.; Dryer, M.; Lisse, C. M.; Dennerl, K.; Zurbuchen, T. H.; Wolk, S. J.; Tielens, A. G. G. M.; Hoekstra, R.

    Aims. We present results of the analysis of cometary X-ray spectra with an extended version of our charge exchange emission model (Bodewits et al. 2006). We have applied this model to the sample of 8 comets thus far observed with the Chandra X-ray observatory and acis spectrometer in the 300 - 1000

  15. Comet P/2001 T3 (Neat)

    Czech Academy of Sciences Publication Activity Database

    Lawrence, K. J.; Pravdo, S. H.; Helin, E. F.; Pravec, Petr; Kušnirák, Peter; Tichá, J.; Tichý, M.; Jelínek, P.

    č. 7733 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  16. Comet P/2001 MD 7 (Linear)

    Czech Academy of Sciences Publication Activity Database

    Blythe, M.; Kotková, Lenka; Marsden, B. G.

    č. 7660 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  17. Comet C/2001 Q6 (Neat)

    Czech Academy of Sciences Publication Activity Database

    Pravdo, S. H.; Helin, E. F.; Lawrence, K. J.; Tichý, M.; Kotková, Lenka; Wolf, M.; Balam, D.; Shelus, P. J.

    č. 7698 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  18. Comet C/2001 A2 (Linear)

    Czech Academy of Sciences Publication Activity Database

    Pravec, Petr; Kotková, Lenka; Tichý, M.; Kočer, M.

    č. 7564 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  19. Comet C/2001 Q1 (Neat)

    Czech Academy of Sciences Publication Activity Database

    Lawrence, K. J.; Helin, E. F.; Pravdo, S. H.; Pravec, Petr; Kušnirák, Peter; Kočer, M.; Spahr, T. B.

    č. 7685 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics

  20. Comet C/2001 A1 (Linear)

    Czech Academy of Sciences Publication Activity Database

    Blythe, M.; Dawson, M.; Kornos, L.; Koleny, P.; Kotková, Lenka; Tichá, J.; Tichý, M.

    č. 7561 (2001), s. 1 ISSN 0081-0304 R&D Projects: GA ČR GA205/99/0255 Institutional research plan: CEZ:AV0Z1003909 Keywords : comets * astrometry Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics


    Energy Technology Data Exchange (ETDEWEB)

    A' Hearn, Michael F.; Feaga, Lori M.; Sunshine, Jessica M.; Besse, Sebastien; Bodewits, Dennis; Farnham, Tony L.; Kelley, Michael S. [Department of Astronomy, University of Maryland, College Park, MD 20742 (United States); Keller, H. Uwe [Institute for Geophysics and Extraterrestrial Physics, Technische Universitaet Braunschweig, D-38106 Braunschweig (Germany); Kawakita, Hideyo [Department of Physics, Kyoto Sangyo University, Kamigamo JP Kita-ku, Kyoto 603-8555 (Japan); Hampton, Donald L. [Geophysical Institute, University of Alaska Fairbanks, 903 Koyukuk Drive, Fairbanks, AK 99775 (United States); Kissel, Jochen [Max-Planck-Institut for Solar System Research, Max-Planck-Strasse 2, D-37191 Katlenburg-Lindau (Germany); Klaasen, Kenneth P.; Yeomans, Donald K. [Jet Propulsion Laboratory/Caltech, Pasadena, CA 91109 (United States); McFadden, Lucy A. [NASA Goddard Space Flight Center, Greenbelt, MD 20771 (United States); Meech, Karen J. [Institute for Astronomy, 2680 Woodlawn Drive, Honolulu, HI 96822 (United States); Schultz, Peter H. [Department of Geological Sciences, Brown University, Providence, RI 02912 (United States); Thomas, Peter C.; Veverka, Joseph [Department of Astronomy, Cornell University, Ithaca, NY 14853 (United States); Groussin, Olivier [Laboratoire d' Astrophysique de Marseille, Universite d' Aix-Marseille and CNRS, UMR7326, 38 rue F. Joliot-Curie, F-13388 Marseille Cedex 13 (France); Lisse, Carey M., E-mail: [Space Department, JHU-APL, 11100 Johns Hopkins Road, Laurel, MD 20723 (United States); and others


    We describe recent results on the CO/CO{sub 2}/H{sub 2}O composition of comets together with a survey of older literature (primarily for CO/H{sub 2}O) and compare these with models of the protoplanetary disk. Even with the currently small sample, there is a wide dispersion in abundance ratios and little if any systematic difference between Jupiter-family comets (JFCs) and long-period and Halley-type comets (LPCs and HTCs). We argue that the cometary observations require reactions on grain surfaces to convert CO to CO{sub 2} and also require formation of all types of comets in largely, but not entirely, overlapping regions, probably between the CO and CO{sub 2} snow lines. Any difference in the regions of formation is in the opposite direction from the classical picture with the JFCs having formed closer to the Sun than the LPCs. In the classical picture, the LPCs formed in the region of the giant planets and the JFCs formed in the Kuiper Belt. However, these data suggest, consistent with suggestions on dynamical grounds, that the JFCs and LPCs formed in largely overlapping regions where the giant planets are today and with JFCs on average forming slightly closer to the Sun than did the LPCs. Presumably at least the JFCs passed through the scattered disk on their way to their present dynamical family.

  2. Comets and the origin and evolution of life

    CERN Document Server

    McKay, Christopher P


    Nine years after the publication of Comets and the Origin and Evolution of Life, one of the pioneering books in Astrobiology, this second edition revisits the role comets may have played in the origins and evolution of life. Recent analyses of Antarctic micrometeorites and ancient rocks in Australia and South Africa, the continuing progress in discovering complex organic macromolecules in comets, protostars and interstellar clouds, new insights into organic synthesis in comets, and numerical simulations of comet impacts on the Earth and other members of the solar system yield a spectacular wea

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


    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.


    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...

  4. Lung Injury; Relates to Real-Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung (United States)


    AWARD NUMBER: W81XWH-16-1-0253 TITLE: Lung Injury; Relates to Real- Time Endoscopic Monitoring of Single Cells Respiratory Health in Lung...response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and...Sep 2016 - 31 Aug 2017 5a. CONTRACT NUMBER 4. TITLE AND SUBTITLE Lung Injury; Relates to Real- Time Endoscopic Monitoring of Single Cells Respiratory

  5. 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


    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

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

    KAUST Repository

    Sakaki, Kelly


    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.

  7. SC3: consensus clustering of single-cell RNA-seq data. (United States)

    Kiselev, Vladimir Yu; Kirschner, Kristina; Schaub, Michael T; Andrews, Tallulah; Yiu, Andrew; Chandra, Tamir; Natarajan, Kedar N; Reik, Wolf; Barahona, Mauricio; Green, Anthony R; Hemberg, Martin


    Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach ( We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

  8. PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol's iodine solution. (United States)

    Henrichs, D W; Renshaw, M A; Santamaria, C A; Richardson, B; Gold, J R; Campbell, L


    A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol's iodine preservative. Destaining of Lugol's-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.

  9. The Greatest Comets in History Broom Stars and Celestial Scimitars

    CERN Document Server

    Seargent, David A J


    Comets have fascinated and awed humankind since ancient times. Of the thousands of comets recorded throughout history, those deemed to have been the most spectacular have been described in the accounts of eyewitnesses and often recorded in official documents. This book introduces you to the greatest of the greats, starting with the comet in 372 B. C. called "Aristotle’s Comet" and ending with the spectacular appearance of McNaught’s Comet in 2007. There is an introductory chapter explaining what comets are and how they are classified, and correcting a few popular misconceptions. Later in the book you will read about the different returns of Halley’s Comet and the Kreutz sungrazing group, often called the kamikaze comets. There is even a chapter on comets that were visible in broad daylight. This book is unique. There are a few books on comets that make passing reference to some of the more famous or spectacular objects of the past, and a few catalogs with long lists of comets. But little detailed and de...

  10. Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer. (United States)

    Liu, Mingshan; Liu, Yang; Di, Jiabo; Su, Zhe; Yang, Hong; Jiang, Beihai; Wang, Zaozao; Zhuang, Meng; Bai, Fan; Su, Xiangqian


    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.

  11. Using single cell sequencing data to model the evolutionary history of a tumor. (United States)

    Kim, Kyung In; Simon, Richard


    The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumor. This may facilitate understanding how key mutations accumulate and evolve in lineages to form a heterogeneous tumor. We provide a computational method to infer an evolutionary mutation tree based on single cell sequencing data. Our approach differs from traditional phylogenetic tree approaches in that our mutation tree directly describes temporal order relationships among mutation sites. Our method also accommodates sequencing errors. Furthermore, we provide a method for estimating the proportion of time from the earliest mutation event of the sample to the most recent common ancestor of the sample of cells. Finally, we discuss current limitations on modeling with single cell sequencing data and possible improvements under those limitations. Inferring the temporal ordering of mutational sites using current single cell sequencing data is a challenge. Our proposed method may help elucidate relationships among key mutations and their role in tumor progression.

  12. Long-period comets with non-gravitational effects (United States)

    Królikowska, M.


    The influence of non-gravitational effects on the motion of long-period comets is analyzed. Investigations were performed for about 60 nearly-parabolic comets discovered during the last thirty years. All three non-gravitational parameters A1, A2, A3 were detected in the motion of 19 comets on the basis of positional observations only. For these comets the standard g(r) function as well as some of its modifications were used to model the ice sublimation from the cometary nucleus. For 14 of 19 comets the orientation of spin axes of the nucleus were also determined. Finally, the magnitude of the non-gravitational effects derived for the long-period comets have been compared with observed the non-gravitational perturbations on periodic comets. Tables 1, 2, 4 and 6 are only available in electronic form at

  13. GelTouch

    DEFF Research Database (Denmark)

    Miruchna, Viktor; Walter, Robert; Lindlbauer, David


    We present GelTouch, a gel-based layer that can selectively transition between soft and stiff to provide tactile multi-touch feedback. It is flexible, transparent when not activated, and contains no mechanical, electromagnetic, or hydraulic components, resulting in a compact form factor (a 2mm thin...... touchscreen layer for our prototype). The activated areas can be morphed freely and continuously, without being limited to fixed, predefined shapes. GelTouch consists of a poly(N-isopropylacrylamide) gel layer which alters its viscoelasticity when activated by applying heat (>32 C). We present three different...

  14. Transport Phenomena in Gel


    Masayuki Tokita


    Gel becomes an important class of soft materials since it can be seen in a wide variety of the chemical and the biological systems. The unique properties of gel arise from the structure, namely, the three-dimensional polymer network that is swollen by a huge amount of solvent. Despite the small volume fraction of the polymer network, which is usually only a few percent or less, gel shows the typical properties that belong to solids such as the elasticity. Gel is, therefore, regarded as a dilu...

  15. Unveiling the formation and evolution of comets (United States)

    Lasue, J.; Levasseur-Regourd, A. C.; Botet, R.; Coradini, A.; Desanctis, M. C.; Kofman, W.


    Comet nuclei are considered as the most pristine bodies of the solar system and consequently their study sheds an important light on the processes occurring during the initial stages of the solar system formation. The analysis of the porosity and bulk density of such primordial bodies is especially important to understand their capacity to retain volatile components (organics and ices) present in the early solar nebula. Typical tensile strengths deduced for comet nuclei range from below 102N.m-2 from the Deep Impact mission [1] up to 104N.m-2 from the study of comet C/1999 S4 LINEAR breakup [2] and meteoroids [3]. A bulk density of about 350 kg/m3 has been obtained for 9P/Tempel 1 from the Deep Impact mission [4]. Moreover the properties of dust released from the comets strongly confirm such values. Instruments flying-by comet 1P/Halley had discovered the presence of organics, and pointed out the dust low albedo and extremely low density while analyses of Interplanetary Dust Particles collected in the stratosphere and remote spectroscopic observations have indicated that cometary dust consists of an un-equilibrated heterogeneous mixture of organic refractory materials and of amorphous and crystalline silicate minerals [5], as recently confirmed by Stardust [6]. Observations of the solar scattered light, together with elaborate simulations, give an estimation of the mass ratio between silicates and absorbing organics, the size distribution and the structure of the dust particles, suggesting that a fair amount consists in fluffy aggregates built up from submicronic grains [7,8], as recently confirmed by the analysis of dust craters and aerogel tracks on Stardust collector showing for some large particles (up to 100 μm) an extraordinary fluffy structure [9]. Simulations have been developed in our teams to describe the aspects of comet aggregation and evolution that have not been thoroughly explained yet. Particle aggregation simulations taking into account cohesive

  16. ESA Unveils Its New Comet Chaser. (United States)


    The objective is to study one of these primordial objects at close quarters by placing a lander on its surface and chasing, with an orbiter, the comet for millions of kilometres through space. Comets - among the oldest (4.6 billion years!) and last altered objects in the solar system - are regarded as the building blocks from which the planets formed. Thus the Rosetta's discoveries will allow the scientists to learn more about birth and evolution of the planets and about the origin of life on the Earth. The final design of the Rosetta orbiter will be revealed for the first time at the Royal Society in London on 1 July when a 1:4 scale model will be unveiled by ESA's Director of Science, Prof.. Roger Bonnet. (The full size version of the spacecraft is 32 metres across, so large that it would stretch the entire width of a football pitch. Almost 90 of this is accounted for by the giant solar panels which are needed to provide electrical power in the dark depths of the Solar System). "Rosetta is a mission of major scientific importance," said Prof. Bonnet. "It will build on the discoveries made by Giotto and confirm ESA's leading role in the exploration of the Solar System and the Universe as a whole." The timing of this event has been chosen to coincide with the London meeting of the Rosetta Science Working Team and the second Earth flyby of the now non-operational Giotto spacecraft. In addition, the opening of the British Museum's 'Cracking Codes' Exhibition, for which the Rosetta Stone is the centrepiece, is set to take place on 10 July. The Rosetta mission. Rosetta is the third Cornerstone in ESA's 'Horizon 2000' long-term scientific programme. It will be launched by Ariane 5 rocket from Kourou spaceport in French Guiana in January 2003. In order to gain sufficient speed to reach the distant comet, Rosetta will require gravity assists from the Earth (twice) and Mars. After swinging around Mars in May 2005, Rosetta will return to Earth's vicinity in October 2005 and

  17. Reducing the Asteroid and Comet Impact Hazard (United States)

    Crawford, David


    Of the 140 impact craters known on the surface of Earth, the most famous was created about 65 million years ago when a 10 km asteroid or comet came down in shallow water near the present day town of Chicxulub, Mexico. With a kinetic energy equivalent to 100 trillion tons of TNT, the impact event lofted enough debris onto globe-straddling trajectories to flash heat much of the surface of the Earth and then darken the skies for several years. Numerous investigations have demonstrated that such an event, which happens, on average, every 100 million years, caused extreme stress on Earth's climate and most likely led to the extinction of many species. Computational simulations demonstrate that more numerous asteroids or comets as small as 1 km in diameter, impacting, on average, every 300,000 years may be globally catastrophic. Indeed, the odds of an individual dying from a relatively frequent 1 km impacting object (about 1 in 10,000) are substantially greater than from the impact of an infrequent dinosaur killer (1 in 1,000,000). What can we do to reduce the hazard from impacting comets and asteroids? First, we should find what's out there with our name on it. Only about 10 percent of the potential Earth-crossing asteroids have been found. Even at the greatly increased detection rate of recent years, it will be several decades before we've found 90 percent of the Earth-crossers. Second, we should learn everything we can about the physical, compositional and mechanical properties of asteroids and comets. A recent computational study demonstrated that weakly bound asteroids (little more than rubble piles) are easier to break than deflect(E. Asphaug, S. J. Ostro, R. S. Hudson, D. J. Scheeres and W. Benz (1998), Nature, Vol. 393, pp. 437-440.). Is this an advantage or disadvantage? Third, we should study potential means of mitigating the hazard by deflecting the object while still in space or evacuating affected regions (such as coastlines) of the Earth. Because the

  18. Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data (United States)

    Kussell, Edo


    Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748

  19. Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data.

    Directory of Open Access Journals (Sweden)

    Takashi Nozoe


    Full Text Available Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells' response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution.

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

    Directory of Open Access Journals (Sweden)

    Liang Huang


    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.

  1. Cyanocobalamin Nasal Gel (United States)

    ... to supply extra vitamin B12 to people who need unusually large amounts of this vitamin because they are pregnant or have certain diseases. ... Cyanocobalamin nasal gel will supply you with enough vitamin B12 only as ... it regularly. You may need to use cyanocobalamin nasal gel every week for ...

  2. Modeling chemoresponsive polymer gels. (United States)

    Kuksenok, Olga; Deb, Debabrata; Dayal, Pratyush; Balazs, Anna C


    Stimuli-responsive gels are vital components in the next generation of smart devices, which can sense and dynamically respond to changes in the local environment and thereby exhibit more autonomous functionality. We describe recently developed computational methods for simulating the properties of such stimuli-responsive gels in the presence of optical, chemical, and thermal gradients. Using these models, we determine how to harness light to drive shape changes and directed motion in spirobenzopyran-containing gels. Focusing on oscillating gels undergoing the Belousov-Zhabotinksy reaction, we demonstrate that these materials can spontaneously form self-rotating assemblies, or pinwheels. Finally, we model temperature-sensitive gels that encompass chemically reactive filaments to optimize the performance of this system as a homeostatic device for regulating temperature. These studies could facilitate the development of soft robots that autonomously interconvert chemical and mechanical energy and thus perform vital functions without the continuous need of external power sources.

  3. Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative. (United States)

    Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi


    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara


    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...... causing significant DNA damage was 20 μM for H2O2 and 200 mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA...

  5. Identification of irradiated pepper with comet assay

    International Nuclear Information System (INIS)

    Prieto Miranda, Enrique Fco.; Moreno Alvarez, Damaris L.; Carro Palacio, Sandra; Iglesia Enriquez, Isora


    The treatment of foods with ionizing radiations is a technological process utilized in order to increase the hygienic quality and the storage time of the foods. Several methods of detection of irradiated foods have been recommended. The comet assay of DNA is one fast and economical technique for the qualitative identification of irradiated foods. The objective of the present paper was to identify with the comet assay technique the modifications of the DNA molecule of irradiated pepper storage at environment and refrigeration temperatures and different post-irradiation times for different absorbed dose values, (0.1, 0.3 and 0.5 kGy). It was demonstrated that for the high absorbed dose values was observed a greater break into fragments of the DNA molecule, which shows the application of this technique for the identification of irradiated foods. (author)

  6. Spatio-temporally controlled transfection by quantitative injection into a single cell. (United States)

    Kwon, Hyosung; Park, Hang-soo; Yu, Jewon; Hong, Sunghoi; Choi, Yeonho


    Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. How to design a single-cell RNA-sequencing experiment: pitfalls, challenges and perspectives. (United States)

    Dal Molin, Alessandra; Di Camillo, Barbara


    The sequencing of the transcriptome of single cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types in heterogeneous cell populations or for the study of stochastic gene expression. In recent years, various experimental methods and computational tools for analysing single-cell RNA-sequencing data have been proposed. However, most of them are tailored to different experimental designs or biological questions, and in many cases, their performance has not been benchmarked yet, thus increasing the difficulty for a researcher to choose the optimal single-cell transcriptome sequencing (scRNA-seq) experiment and analysis workflow. In this review, we aim to provide an overview of the current available experimental and computational methods developed to handle single-cell RNA-sequencing data and, based on their peculiarities, we suggest possible analysis frameworks depending on specific experimental designs. Together, we propose an evaluation of challenges and open questions and future perspectives in the field. In particular, we go through the different steps of scRNA-seq experimental protocols such as cell isolation, messenger RNA capture, reverse transcription, amplification and use of quantitative standards such as spike-ins and Unique Molecular Identifiers (UMIs). We then analyse the current methodological challenges related to preprocessing, alignment, quantification, normalization, batch effect correction and methods to control for confounding effects. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  8. Polydimethylsiloxane (PDMS Sub-Micron Traps for Single-Cell Analysis of Bacteria

    Directory of Open Access Journals (Sweden)

    Dietrich Kohlheyer


    Full Text Available Microfluidics has become an essential tool in single-cell analysis assays for gaining more accurate insights into cell behavior. Various microfluidics methods have been introduced facilitating single-cell analysis of a broad range of cell types. However, the study of prokaryotic cells such as Escherichia coli and others still faces the challenge of achieving proper single-cell immobilization simply due to their small size and often fast growth rates. Recently, new approaches were presented to investigate bacteria growing in monolayers and single-cell tracks under environmental control. This allows for high-resolution time-lapse observation of cell proliferation, cell morphology and fluorescence-coupled bioreporters. Inside microcolonies, interactions between nearby cells are likely and may cause interference during perturbation studies. In this paper, we present a microfluidic device containing hundred sub-micron sized trapping barrier structures for single E. coli cells. Descendant cells are rapidly washed away as well as components secreted by growing cells. Experiments show excellent growth rates, indicating high cell viability. Analyses of elongation and growth rates as well as morphology were successfully performed. This device will find application in prokaryotic single-cell studies under constant environment where by-product interference is undesired.

  9. A modified single-cell electroporation method for molecule delivery into a motile protist, Euglena gracilis. (United States)

    Ohmachi, Masashi; Fujiwara, Yoshie; Muramatsu, Shuki; Yamada, Koji; Iwata, Osamu; Suzuki, Kengo; Wang, Dan Ohtan


    Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media. (United States)

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


    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).

  11. Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing. (United States)

    Mora-Castilla, Sergio; To, Cuong; Vaezeslami, Soheila; Morey, Robert; Srinivasan, Srimeenakshi; Dumdie, Jennifer N; Cook-Andersen, Heidi; Jenkins, Joby; Laurent, Louise C


    As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing. © 2016 Society for Laboratory Automation and Screening.

  12. visnormsc: A Graphical User Interface to Normalize Single-cell RNA Sequencing Data. (United States)

    Tang, Lijun; Zhou, Nan


    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 . 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.

  13. Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome (United States)

    Teschendorff, Andrew E.; Enver, Tariq


    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.

  14. Ultrasound lung comets in extravascular fluid diagnostics

    Directory of Open Access Journals (Sweden)

    М. Н. Алехин


    Full Text Available The review is devoted to the analysis of ultrasound lung comets (ULCs that may be observed during ultrasound lung examination in cardiac patients in the case of accumulation of extravascular fluid in the lungs. Recent data on the mechanisms of these artefacts, examination and outcome evaluation methods are discussed. Differential diagnostics problems in patients with cardiac and pulmonary genesis of ULCs are also covered.

  15. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq). (United States)

    Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin


    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.


    Energy Technology Data Exchange (ETDEWEB)

    Davis, M.; Hut, P.; Muller, R.A.


    A 26-Myr periodicity has recently been seen in the fossil record of extinction in the geological past. At least two of these extinctions are known to be associated with the impact on the Earth of a comet or asteroid with a diameter of a few kilometres. We propose that the periodic events are triggered by an unseen companion to the Sun, travelling in a moderately eccentric orbit, which at its closest approach (perihelion) passes through the 'Oort cloud' of comets which surrounds the Sun. During each passage this unseen solar companion perturbs the orbits of these comets, sending a large number of them (over 1 x 10{sup 9}) into paths which reach the inner Solar System. Several of these hit the Earth, on average, in the following million years. At present the unseen companion should be approximately at its maximum distance from the Sun, {approx}2.4 light yr, and it will present no danger to the Earth until approximately AD 15,000,000.

  17. Comets: Role and importance to exobiology (United States)

    Delsemme, Armand H.


    The transfer of organic compounds from interstellar space to the outskirts of a protoplanetary disk, their accretion into cometary objects, and the transport of the latter into the inner solar system by orbital diffusion throw a new light on the central problem of exobiology. It suggests the existence of a cosmic mechanism, working everywhere, that can supply prebiotic compounds to ubiquitous rocky planets, in search of the proper environment to start life in many places in the Universe. Under the heading of chemistry of the cometary nucleus, the following topics are covered: radial homogeneity of the nucleus; the dust-to-ice ratio; nature of the dust grains; origin of the dust in comets; nature of the volatile fraction; the CO distribution in comet Halley; dust contribution to the volatile fraction; elemental balance sheet of comet Halley; quantitative molecular analysis of the volatile fraction; and isotopic ratios. Under the heading of exogenous origin of carbon on terrestrial planets the following topics are covered: evidence for a high-temperature phase; from planetesimals to planets; a veneer of volatile and organic material; and cometary contribution.

  18. Perihelion evolution of observed new comets implies the dominance of the galactic tide in making Oort cloud comets discernable (United States)

    Matese, John J.; Lissauer, Jack J.


    For an Oort cloud comet to be seen as a new comet, its perihelion must be moved from a point exterior to the loss cylinder boundary to a point interior to observable limits in a single orbit. The galactic tide can do this continuously, in a non-impulsive manner. Near-parabolic comets, with specific angular momentum H∝ √q, will most easily be made observable. Therefore, to reduce the perihelion distance H must decrease. Since weakly perturbed comets are, in general, more numerous than strongly perturbed comets, we can anticipate that new comets made observable by a weak tidal torque will more likely be first observed when their slowly changing perihelion distances are approaching their minimum osculating values under the action of the tide, rather than receding from their minimum values. That is, defining Δ Htide as the vector change due to the galactic tidal torque during the prior orbit, and Hobs as the observed vector, the sign S≡Sign( Hobs·Δ Htide) will more likely be -1 than +1 if a weak galactic tidal perturbation indeed dominates in making comets observable. Using comet data of the highest quality class (1A) for new comets ( a>10,000 AU), we find that 49 comets have S=-1 and 22 have S=+1. The binomial probability that as many or more would exhibit this characteristic if in fact S=∓1 were equally likely is only 0.0009. This characteristic also persists in other long-period comet populations, lending support to the notion that they are dominated by comets recently arrived from the outer Oort cloud. The preponderance of S=-1 also correlates with weakly perturbed (i.e., smaller semimajor axis) new comets in a statistically significant manner. This is strong evidence that the data are of sufficiently high quality and sufficiently free of observational selection effects to detect this unique imprint of the tide.

  19. Assessment of individual radiosensitivity in human lymphocytes using micronucleus and microgel electrophoresis Comet assays

    Energy Technology Data Exchange (ETDEWEB)

    Giorgio, M. di; Sardi, M.; Busto, M.; Vallerga, M.; Taja, M.; Mairal, I.


    Background and purpose: Individual radiosensitivity is an inherent characteristic, associated with an increased reaction to ionizing radiation on the human body. Individuals show marked differences in radiation sensitivity, which has consequences in the fields of both radiation protection and radiation therapy. It is suggested that DNA repair mechanisms are involved. Consequently, the characterization of DNA repair in lymphocytes through cytokinesis blocked micronucleus (MN) and alkaline single-cell microgel electrophoresis (comet) assays could be suitable approaches to evaluate individual radiosensitivity in vitro. The amins of this study were: 1) to assess the in vitro radisensitivity of peripheral blood lymphocytes from two with the observed clinical response and 2) to test the predictive potential of both techniques. Materials and methods: 38 cancer patients receiving radiation therapy were enrolled in this study. The tumor sites were: head and neck (n=25) and cervic (n=13). 19 pateints were evaluated prior, mid-way and on completion of treatment (prospective group) and 19 patients were evaluated about 2-480 month after radiotherapy (retrospective group). Cytogenetic data from the prospective group were analyzed using a mathematical model to evaluate the attenuation of the cytogenetic effect as a function of the time between a single exposure and blood sampling, estimating a cytogentic recovery factor k. In the retrospective group, blood samples were irradiated in vitro with 0 (control) or 2 Gy and evaluated using MN test. Cytogenetic data were analyzed comparing expected MN frequencies (calibration curve from health donors) with values observed after in vitro irradiation. One over-reactor ad patients that did not develop late effects were also evaluated through comet assay. DNA damage and repair capacity were quantified by the Olive tail moment. Lymphocytes of health individuals were used as reference sample. In the prospective evaluation, factor K correlated

  20. Assessment of individual radiosensitivity in human lymphocytes using micronucleus and microgel electrophoresis Comet assays

    International Nuclear Information System (INIS)

    Giorgio, M. di; Sardi, M.; Busto, M.; Vallerga, M.; Taja, M.; Mairal, I.


    Background and purpose: Individual radiosensitivity is an inherent characteristic, associated with an increased reaction to ionizing radiation on the human body. Individuals show marked differences in radiation sensitivity, which has consequences in the fields of both radiation protection and radiation therapy. It is suggested that DNA repair mechanisms are involved. Consequently, the characterization of DNA repair in lymphocytes through cytokinesis blocked micronucleus (MN) and alkaline single-cell microgel electrophoresis (comet) assays could be suitable approaches to evaluate individual radiosensitivity in vitro. The amins of this study were: 1) to assess the in vitro radisensitivity of peripheral blood lymphocytes from two with the observed clinical response and 2) to test the predictive potential of both techniques. Materials and methods: 38 cancer patients receiving radiation therapy were enrolled in this study. The tumor sites were: head and neck (n=25) and cervic (n=13). 19 pateints were evaluated prior, mid-way and on completion of treatment (prospective group) and 19 patients were evaluated about 2-480 month after radiotherapy (retrospective group). Cytogenetic data from the prospective group were analyzed using a mathematical model to evaluate the attenuation of the cytogenetic effect as a function of the time between a single exposure and blood sampling, estimating a cytogentic recovery factor k. In the retrospective group, blood samples were irradiated in vitro with 0 (control) or 2 Gy and evaluated using MN test. Cytogenetic data were analyzed comparing expected MN frequencies (calibration curve from health donors) with values observed after in vitro irradiation. One over-reactor ad patients that did not develop late effects were also evaluated through comet assay. DNA damage and repair capacity were quantified by the Olive tail moment. Lymphocytes of health individuals were used as reference sample. In the prospective evaluation, factor K correlated

  1. Comparison of comet 81P/Wild 2 dust with interplanetary dust from comets. (United States)

    Ishii, Hope A; Bradley, John P; Dai, Zu Rong; Chi, Miaofang; Kearsley, Anton T; Burchell, Mark J; Browning, Nigel D; Molster, Frank


    The Stardust mission returned the first sample of a known outer solar system body, comet 81P/Wild 2, to Earth. The sample was expected to resemble chondritic porous interplanetary dust particles because many, and possibly all, such particles are derived from comets. Here, we report that the most abundant and most recognizable silicate materials in chondritic porous interplanetary dust particles appear to be absent from the returned sample, indicating that indigenous outer nebula material is probably rare in 81P/Wild 2. Instead, the sample resembles chondritic meteorites from the asteroid belt, composed mostly of inner solar nebula materials. This surprising finding emphasizes the petrogenetic continuum between comets and asteroids and elevates the astrophysical importance of stratospheric chondritic porous interplanetary dust particles as a precious source of the most cosmically primitive astromaterials.

  2. An automated system for high-throughput single cell-based breeding (United States)

    Yoshimoto, Nobuo; Kida, Akiko; Jie, Xu; Kurokawa, Masaya; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D.; Nikaido, Itoshi; Ueda, Hiroki R.; Tatematsu, Kenji; Tanizawa, Katsuyuki; Kondo, Akihiko; Fujii, Ikuo; Kuroda, Shun'ichi


    When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms. PMID:23378922

  3. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software. (United States)

    Kaiser, Matthias; Jug, Florian; Julou, Thomas; Deshpande, Siddharth; Pfohl, Thomas; Silander, Olin K; Myers, Gene; van Nimwegen, Erik


    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 analysis software that enable long-term quantitative tracking of growth and gene expression in single cells. The dual-input Mother Machine (DIMM) chip enables controlled and continuous variation of external conditions, allowing direct observation of gene regulatory responses to changing conditions in single cells. The Mother Machine Analyzer (MoMA) software achieves unprecedented accuracy in segmenting and tracking cells, and streamlines high-throughput curation with a novel leveraged editing procedure. We demonstrate the power of the method by uncovering several novel features of an iconic gene regulatory program: the induction of Escherichia coli's lac operon in response to a switch from glucose to lactose.

  4. Single-cell RNA-sequencing: The future of genome biology is now. (United States)

    Picelli, Simone


    Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis.

  5. DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning. (United States)

    Angermueller, Christof; Lee, Heather J; Reik, Wolf; Stegle, Oliver


    Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells. We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variability.

  6. Comparison of whole genome amplification techniques for human single cell exome sequencing. (United States)

    Borgström, Erik; Paterlini, Marta; Mold, Jeff E; Frisen, Jonas; Lundeberg, Joakim


    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.

  7. The asteroid-comet continuum from laboratory and space analyses of comet samples and micrometeorites (United States)

    Engrand, Cecile; Duprat, Jean; Bardin, Noemie; Dartois, Emmanuel; Leroux, Hugues; Quirico, Eric; Benzerara, Karim; Rémusat, Laurent; Dobrică, Elena; Delauche, Lucie; Bradley, John; Ishii, Hope; Hilchenbach, Martin; COSIMA Team


    Comets are probably the best archives of the nascent solar system, 4.5 Gyr ago, and their compositions reveal crucial clues on the structure and dynamics of the early protoplanetary disk. Anhydrous minerals (olivine and pyroxene) have been identified in cometary dust for a few decades. Surprisingly, samples from comet Wild2 returned by the Stardust mission in 2006 also contain high temperature mineral assemblages like chondrules and refractory inclusions, which are typical components of primitive meteorites (carbonaceous chondrites - CCs). A few Stardust samples have also preserved some organic matter of comet Wild 2 that share some similarities with CCs. Interplanetary dust falling on Earth originate from comets and asteroids in proportions to be further constrained. These cosmic dust particles mostly show similarities with CCs, which in turn only represent a few percent of meteorites recovered on Earth. At least two (rare) families of cosmic dust particles have shown strong evidences for a cometary origin: the chondritic porous interplanetary dust particles (CP-IDPs) collected in the terrestrial stratosphere by NASA, and the ultracarbonaceous Antarctic Micrometeorites (UCAMMs) collected from polar snow and ice by French and Japanese teams. The Rosetta mission currently carries dust analyzers capable of measuring dust flux, sizes, physical properties and compositions of dust particles from the Jupiter family comet 67P/Churyumov-Gerasimenko (COSIMA, GIADA, MIDAS), as well as gas analyzers (ROSINA, PTOLEMY, COSAC). A growing number of evidences highlights the existence of a continuum between asteroids and comets, already in the early history of the solar system. We will present the implications of the analyses of samples in the laboratory and in space to a better understanding of the early protoplanetary disk.

  8. The persistent coma of Comet P/Schwassmann-Wachmann 1

    International Nuclear Information System (INIS)

    Jewitt, D.


    Time-series photometry of Comet P/Schwassmann-Wachmann 1 in both 1987 and 1988 shows that this comet is continually active despite its large heliocentric distance. The observed activity, upon which the famous outbursts of this comet are superposed, may be driven by the sublimation of crystalline water ice at the nucleus surface. A simple model which accounts for both the continuous activity and the sporadic outbursts is suggested. 34 refs

  9. Reservoirs for Comets: Compositional Differences Based on Infrared Observations (United States)

    Disanti, Michael A.; Mumma, Michael J.

    Tracing measured compositions of comets to their origins continues to be of keen interest to cometary scientists and to dynamical modelers of Solar System formation and evolution. This requires building a taxonomy of comets from both present-day dynamical reservoirs: the Kuiper Belt (hereafter KB), sampled through observation of ecliptic comets (primarily Jupiter Family comets, or JFCs), and the Oort cloud (OC), represented observationally by the long-period comets and by Halley Family comets (HFCs). Because of their short orbital periods, JFCs are subjected to more frequent exposure to solar radiation compared with OC comets. The recent apparitions of the JFCs 9P/Tempel 1 and 73P/Schwassmann-Wachmann 3 permitted detailed observations of material issuing from below their surfaces—these comets added significantly to the compositional database on this dynamical class, which is under-represented in studies of cometary parent volatiles. This chapter reviews the latest techniques developed for analysis of high-resolution spectral observations from ˜2-5 μm, and compares measured abundances of native ices among comets. While no clear compositional delineation can be drawn along dynamical lines, interesting comparisons can be made. The sub-surface composition of comet 9P, as revealed by the Deep Impact ejecta, was similar to the majority of OC comets studied. Meanwhile, 73P was depleted in all native ices except HCN, similar to the disintegrated OC comet C/1999 S4 (LINEAR). These results suggest that 73P may have formed in the inner giant planets' region while 9P formed farther out or, alternatively, that both JFCs formed farther from the Sun but with 73P forming later in time.

  10. Studying Short-Period Comets and Long-Period Comets Detected by WISE/NEOWISE (United States)

    Kramer, Emily A.; Fernández, Yanga R.; Bauer, James M.; Stevenson, Rachel; Mainzer, Amy K.; Grav, Tommy; Masiero, Joseph; Walker, Russell G.; Lisse, Carey M.


    The Wide-field Infrared Survey Explorer (WISE) mission surveyed the sky in four infrared wavelength bands (3.4, 4.6, 12 and 22-micron) between January 2010 and February 2011 [1, 2]. During the mission, WISE serendipitously observed 160 comets, including 21 newly discovered objects. About 89 of the comets observed by WISE displayed a significant dust tail in the 12 and 22-micron (thermal emission) bands, showing a wide range of activity levels and dust morphology. Since the observed objects are a mix of both long-period comets (LPCs) and short-period comets (SPCs), differences in their activity can be used to better understand the thermal evolution that each of these populations has undergone. For the comets that displayed a significant dust tail, we have estimated the sizes and ages of the particles using dynamical models based on the Finson-Probstein method [3, 4]. For a selection of 40 comets, we have then compared these models to the data using a novel tail-fitting method that allows the best-fit model to be chosen analytically rather than subjectively. For comets that were observed multiple times by WISE, the dust tail particle properties were estimated separately, and then compared. We find that the dust tails of both LPCs and SPCs are primarily comprised of ~mm to cm sized particles, which were the result of emission that occurred several months to several years prior to the observations. The LPCs nearly all have strong dust emission close to the comet's perihelion distance, and the SPCs mostly have strong dust emission close to perihelion, but some have strong dust emission well before perihelion. Acknowledgments: This publication makes use of data products from (1) WISE, which is a joint project of UCLA and JPL/Caltech, funded by NASA; and (2) NEOWISE, which is a project of JPL/Caltech, funded by the Planetary Science Division of NASA. EK was supported by a NASA Earth and Space Sciences Fellowship. RS gratefully acknowledges support from the NASA

  11. Comet 81P/wild 2 under a microscope (United States)

    Brownlee, D.; Tsou, P.; Aleon, J.; O'D, Alexander; Araki, T.; Bajt, S.; Baratta, G.A.; Bastien, R.; Bland, P.; Bleuet, P.; Borg, J.; Bradley, J.P.; Brearley, A.; Brenker, F.; Brennan, S.; Bridges, J.C.; Browning, N.D.; Brucato, J.R.; Bullock, E.; Burchell, M.J.; Busemann, H.; Butterworth, Anna L.; Chaussidon, M.; Cheuvront, A.; Chi, M.; Cintala, M.J.; Clark, B. C.; Clemett, S.J.; Cody, G.; Colangeli, L.; Cooper, G.; Cordier, P.; Daghlian, C.; Dai, Z.; D'Hendecourt, L.; Djouadi, Z.; Dominguez, G.; Duxbury, T.; Dworkin, J.P.; Ebel, D.S.; Economou, T.E.; Fakra, S.; Fairey, S.A.J.; Fallon, S.; Ferrini, G.; Ferroir, T.; Fleckenstein, H.; Floss, C.; Flynn, G.; Franchi, I.A.; Fries, M.; Gainsforth, Z.; Gallien, J.-P.; Genge, M.; Gilles, M.K.; Gillet, P.; Gilmour, J.; Glavin, D.P.; Gounelle, M.; Grady, M.M.; Graham, G.A.; Grant, P.G.; Green, S.F.; Grossemy, F.; Grossman, L.; Grossman, J.N.; Guan, Y.; Hagiya, K.; Harvey, R.; Heck, P.; Herzog, G.F.; Hoppe, P.; Horz, F.; Huth, J.; Hutcheon, I.D.; Ignatyev, K.; Ishii, H.; Ito, M.; Jacob, D.; Jacobsen, C.; Jacobsen, S.; Jones, S.; Joswiak, D.; Jurewicz, A.; Kearsley, A.T.; Keller, L.P.; Khodja, H.; Kilcoyne, A.L.D.; Kissel, J.; Krot, A.; Langenhorst, F.; Lanzirotti, A.; Le, L.; Leshin, L.A.; Leitner, J.; Lemelle, L.; Leroux, H.; Liu, M.-C.; Luening, K.; Lyon, I.; MacPherson, G.; Marcus, M.A.; Marhas, K.; Marty, B.; Matrajt, G.; McKeegan, K.; Meibom, A.; Mennella, V.; Messenger, K.; Messenger, S.; Mikouchi, T.; Mostefaoui, S.; Nakamura, T.; Nakano, T.; Newville, M.; Nittler, L.R.; Ohnishi, I.; Ohsumi, K.; Okudaira, K.; Papanastassiou, D.A.; Palma, R.; Palumbo, M.E.; Pepin, R.O.; Perkins, D.; Perronnet, M.; Pianetta, P.; Rao, W.; Rietmeijer, F.J.M.; Robert, F.; Rost, D.; Rotundi, A.; Ryan, R.; Sandford, S.A.; Schwandt, C.S.; See, T.H.; Schlutter, D.; Sheffield-Parker, J.; Simionovici, A.; Simon, S.; Sitnitsky, I.; Snead, C.J.; Spencer, M.K.; Stadermann, F.J.; Steele, A.; Stephan, T.


    The Stardust spacecraft collected thousands of particles from comet 81P/Wild 2 and returned them to Earth for laboratory study. The preliminary examination of these samples shows that the nonvolatile portion of the comet is an unequilibrated assortment of materials that have both presolar and solar system origin. The comet contains an abundance of silicate grains that are much larger than predictions of interstellar grain models, and many of these are high-temperature minerals that appear to have formed in the inner regions of the solar nebula. Their presence in a comet proves that the formation of the solar system included mixing on the grandest scales.

  12. X-rays from comets - a surprising discovery

    CERN Multimedia

    CERN. Geneva


    Comets are kilometre-size aggregates of ice and dust, which remained from the formation of the solar system. It was not obvious to expect X-ray emission from such objects. Nevertheless, when comet Hyakutake (C/1996 B2) was observed with the ROSAT X-ray satellite during its close approach to Earth in March 1996, bright X-ray emission from this comet was discovered. This finding triggered a search in archival ROSAT data for comets, which might have accidentally crossed the field of view during observations of unrelated targets. To increase the surprise even more, X-ray emission was detected from four additional comets, which were optically 300 to 30 000 times fainter than Hyakutake. For one of them, comet Arai (C/1991 A2), X-ray emission was even found in data which were taken six weeks before the comet was optically discovered. These findings showed that comets represent a new class of celestial X-ray sources. The subsequent detection of X-ray emission from several other comets in dedicated observations confir...

  13. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    Directory of Open Access Journals (Sweden)

    Satomi Kawaguchi


    Full Text Available Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test. WTK1 human lymphoblastoid cells were exposed to methyl nitrosourea (MNU, ethyl nitrosourea (ENU, methyl methanesulfonate (MMS, ethyl methanesulfonate (EMS, bleomycin (BLM, or UVC. In Comet assay, cells were exposed to each mutagen with (Comet assay/araC and without (Comet assay DNA repair inhibitors (araC and hydroxyurea. Furthermore, acellular Comet assay (acellular assay was performed to determine how single-strand breaks (SSBs as the initial damage contributes to DNA migration and/or to micronucleus formation. The lowest genotoxic dose (LGD, which is defined as the lowest dose at which each mutagen causes a positive response on each genotoxicity assay, was used to compare the power of the Comet assay to detect a low level of genotoxic potential and that of MN test; that is, a low LGD indicates a high power. Results are summarized as follows: (1 for all mutagens studied, LGDs were MN test ≦ Comet assay; (2 except for BLM, LGDs were Comet assay/araC ≦ MN test; (3 except for UVC and MNU, LGDs were acellular assay ≦ Comet assay/araC ≦ MN test ≦ Comet assay. The following is suggested by the present findings: (1 LGD in the Comet assay is higher than that in MN test, which suggests that the power of the MN test to detect a low level of genotoxic potential is superior to that of the Comet assay; (2 for the studied mutagens, all assays were able to detect all mutagens correctly, which suggests that the sensitivity of the Comet assay and that of the MN test were

  14. Human organomics: a fresh approach to understanding human development using single-cell transcriptomics. (United States)

    Camp, J Gray; Treutlein, Barbara


    Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. © 2017. Published by The Company of Biologists Ltd.

  15. All-in-polymer injection molded device for single cell capture using multilevel silicon master fabrication

    DEFF Research Database (Denmark)

    Tanzi, S.; Larsen, S.T.; Matteucci, M.


    This work demonstrates a novel all-in-polymer device for single cell capture applicable for biological recordings. The chip is injection molded and comprises a "cornered" (non planar) aperture. It has been demonstrated how cornered apertures are straightforward to mold in PDMS [1,2]. In this stud...... defects during demolding. Capturing of single PC12 cells has been demonstrated.......This work demonstrates a novel all-in-polymer device for single cell capture applicable for biological recordings. The chip is injection molded and comprises a "cornered" (non planar) aperture. It has been demonstrated how cornered apertures are straightforward to mold in PDMS [1,2]. In this study...

  16. Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

    Directory of Open Access Journals (Sweden)

    Bragheri Francesca


    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.

  17. 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


    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...

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

    DEFF Research Database (Denmark)

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

    Bacteria initiate attachment to the surfaces with the aid of different extracellular polymers. To quantitatively study how these polymers mediate bacterial adhesion and possibly their interactions, it is essential to go down to single cell level, with in mind that cell-to-cell variation should...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live...

  19. Evaluation through comet assay of DNA damage induced in human lymphocytes by alpha particles. Comparison with protons and Co-60 gamma rays

    International Nuclear Information System (INIS)

    Di Giorgio, M.; Kreiner, A. J.; Schuff, J. A.; Vallerga, M. B.; Taja, M. R.; Lopez, F. O.; Alvarez, D. E.; Saint Martin, G.; Burton, A.; Debray, M. E.; Kesque, J. M.; Somacal, H.; Stoliar, P.; Valda, A.; Davidson, J.; Davidson, M.; Ozafran, M. J.; Vazquez, M. E.


    Several techniques with different sensitivity to single-strand breaks and/or double strand breaks were applied to detect DNA breaks generated by high LET particles. Tests that assess DNA damage in single cells might be the appropriate tool to estimate damage induced by particles, facilitating the assessment of heterogeneity of damage in a cell population. The microgel electrophoresis (comet) assay is a sensitive method for measuring DNA damage in single cells. The objective of this work was to evaluate the proficiency of comet assay to assess the effect of high LET radiation on peripheral blood lymphocytes, compared to protons and Co-60 gamma rays. Materials and methods: Irradiations of blood samples were performed at TANDAR laboratory (Argentina). Thin samples of human peripheral blood were irradiated with different doses (0-2.5 Gy) of 20.2 MeV helium-4 particles in the track segment mode, at nearly constant LET. Data obtained were compared with the effect induced by a MeV protons and Co-60 gamma rays. Alkaline comet assay was applied. Comets were quantified by the Olive tail moment. Distribution of the helium-4 particle and protons were evaluated considering Poisson distribution in lymphocyte nuclei. The mean dose per nucleus per particle result 0.053 Gy for protons and 0.178 Gy for helium-4 particles. When cells are exposed to a dose of 0.1 Gy, the hit probability model predicts that 43% of the nuclei should have experienced and alpha traversal while with protons, 85% of the nuclei should be hit. The experimental results show a biphasic response for helium-4 particles (0.1 Gy), indicating the existence of two subpopulations: unhit and hit. Distributions of tail moment as a function of fluence and experimental dose for comets induced by helium-4 particles, protons and Co-60 gamma rays were analyzed. With helium-4 irradiations, lymphocyte nuclei show an Olive tail moment distribution flattened to higher tail moments a dose increase. However, for irradiations with

  20. Periodic mesoporous silica gels

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, M.T.; Martin, J.E.; Odinek, J.G. [and others


    We have synthesized monolithic particulate gels of periodic mesoporous silica by adding tetramethoxysilane to a homogeneous alkaline micellar precursor solution. The gels exhibit 5 characteristic length scales over 4 orders of magnitude: fractal domains larger than the particle size (>500 nm), particles that are {approximately}150 to 500 nm in diameter, interparticle pores that are on the order of the particle size, a feature in the gas adsorption measurements that indicates pores {approximately}10-50 nm, and periodic hexagonal arrays of {approximately}3 nm channels within each particle. The wet gel monoliths exhibit calculated densities as low as {approximately}0.02 g/cc; the dried and calcined gels have bulk densities that range from {approximately}0.3-0.5 g/cc. The materials possess large interparticle ({approximately}1.0-2.3 cc/g) and intraparticle ({approximately}0.6 cc/g) porosities.

  1. Gel purification of RNA. (United States)

    Nilsen, Timothy W


    For many applications, including size selection of RNAs and purification of in vitro transcription products, it is necessary to purify RNAs on a denaturing gel. This procedure describes how to purify transcripts that have been synthesized in vitro. It is useful for labeled or unlabeled RNAs when sufficient mass is present. It can also be used to isolate small RNAs. In general, RNA purification by denaturing gel electrophoresis is practical only when the size of the desired RNA is 600 nucleotides or less.

  2. Conformance Improvement Using Gels

    Energy Technology Data Exchange (ETDEWEB)

    Seright, Randall S.; Schrader, Richard; II Hagstrom, John; Wang, Ying; Al-Dahfeeri, Abdullah; Gary, Raven; Marin; Amaury; Lindquist, Brent


    This research project had two objectives. The first objective was to identify gel compositions and conditions that substantially reduce flow through fractures that allow direct channeling between wells, while leaving secondary fractures open so that high fluid injection and production rates can be maintained. The second objective was to optimize treatments in fractured production wells, where the gel must reduce permeability to water much more than that to oil.

  3. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš; Mukherjee, A.; Velemínský, Jiří


    Roč. 605, 1-2 (2006), s. 17-21 ISSN 1383-5718 R&D Projects: GA ČR GA521/05/0500 Institutional research plan: CEZ:AV0Z50380511 Keywords : DNA migration * Nicotiana tabacum * Single-cell gel electrophoresis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.122, year: 2006

  4. SmashCell: A software framework for the analysis of single-cell amplified genome sequences

    DEFF Research Database (Denmark)

    Harrington, Eoghan D; Arumugam, Manimozhiyan; Raes, Jeroen


    SUMMARY: Recent advances in single-cell manipulation technology, whole genome amplification and high-throughput sequencing have now made it possible to sequence the genome of an individual cell. The bioinformatic analysis of these genomes however is far more complicated than the analysis of those...

  5. Raman spectral dynamics of single cells in the early stages of growth factor stimulation. (United States)

    Takanezawa, Sota; Morita, Shin-ichi; Ozaki, Yukihiro; Sako, Yasushi


    Cell fates change dynamically in response to various extracellular signals, including growth factors that stimulate differentiation and proliferation. The processes underlying cell-fate decisions are complex and often include large cell-to-cell variations, even within a clonal population in the same environment. To understand the origins of these cell-to-cell variations, we must detect the internal dynamics of single cells that reflect their changing chemical milieu. In this study, we used the Raman spectra of single cells to trace their internal dynamics during the early stages of growth factor stimulation. This method allows nondestructive and inclusive time-series analyses of chemical compositions of the same single cells. Applying a Gaussian mixture model to the major principal components of the single-cell Raman spectra, we detected the dynamics of the chemical states in MCF-7 cancer-derived cells in the absence and presence of differentiation and proliferation factors. The dynamics displayed characteristic variations according to the functions of the growth factors. In the differentiation pathway, the chemical composition changed directionally between multiple states, including both reversible and irreversible state transitions. In contrast, in the proliferation pathway, the chemical composition was homogenized into a single state. The differentiation factor also stimulated fluctuations in the chemical composition, whereas the proliferation factor did not. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Optimization and Modeling of Quadrupole Orbitrap Parameters for Sensitive Analysis toward Single-Cell Proteomics. (United States)

    Sun, Bingyun; Kovatch, Jessica Rae; Badiong, Albert; Merbouh, Nabyl


    Single-cell proteomics represents a field of extremely sensitive proteomic analysis, owing to the minute amount of yet complex proteins in a single cell. Without amplification potential as of nucleic acids, single-cell mass spectrometry (MS) analysis demands special instrumentation running with optimized parameters to maximize the sensitivity and throughput for comprehensive proteomic discovery. To facilitate such analysis, we here investigated two factors critical to peptide sequencing and protein detection in shotgun proteomics, i.e. precursor ion isolation window (IW) and maximum precursor ion injection time (ITmax), on an ultrahigh-field quadrupole Orbitrap (Q-Exactive HF). Counterintuitive to the frequently used proteomic parameters for bulk samples (>100 ng), our experimental data and subsequent modeling suggested a universally optimal IW of 4.0 Th for sample quantity ranging from 100 ng to 1 ng, and a sample-quantity dependent ITmax of more than 250 ms for 1-ng samples. Compared with the benchmark condition of IW = 2.0 Th and ITmax = 50 ms, our optimization generated up to 300% increase to the detected protein groups for 1-ng samples. The additionally identified proteins allowed deeper penetration of proteome for better revealing crucial cellular functions such as signaling and cell adhesion. We hope this effort can prompt single-cell and trace proteomic analysis and enable a rational selection of MS parameters.

  7. Obesity modulates inflammation and lipid metabolism oocyte gene expression: A single cell transcriptome perspective (United States)

    This study aimed to compare oocyte gene expression profiles and follicular fluid (FF) content from overweight/obese (OW) women and normal weight (NW) women who were undergoing fertility treatments. Using single cell transcriptomic analyses, we investigated oocyte gene expression using RNA-seq. Serum...

  8. On-slide detection of enzymatic activities in selected single cells

    DEFF Research Database (Denmark)

    Keller, Josephine Geertsen; Tesauro, Cinzia; Coletta, Andrea


    With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in si...

  9. Parallel single cell analysis on an integrated microfluidic platform for cell trapping, lysis and analysis

    NARCIS (Netherlands)

    le Gac, Severine; de Boer, Hans L.; Wijnperle, Daniël; Meuleman, W.; Carlen, Edwin; van den Berg, Albert; Kim, Tae Song; Lee, Yoon-Sik; Chung, Taek-Dong; Jeon, Noo Li; Lee, Sang-Hoon; Suh, Kahp-Yang; Choo, Jaebum; Kim, Yong-Kweon


    We report here a novel and easily scalable microfluidic platform for the parallel analysis of hundreds of individual cells, with controlled single cell trapping, followed by their lysis and subsequent retrieval of the cellular content for on-chip analysis. The device consists of a main channel and

  10. 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


    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

  11. Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

    Directory of Open Access Journals (Sweden)

    Nikolay A. Maslov


    Full Text Available Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-mm-diameter microwells (91.45% was higher than that in 20-mm-diameter microwells (83.19% at an injection flow rate of 2.8 mL/min. However, most of the occupied 20-mm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis.

  12. Single-Cell Memory Regulates a Neural Circuit for Sensory Behavior. (United States)

    Kobayashi, Kyogo; Nakano, Shunji; Amano, Mutsuki; Tsuboi, Daisuke; Nishioka, Tomoki; Ikeda, Shingo; Yokoyama, Genta; Kaibuchi, Kozo; Mori, Ikue


    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.

  13. DeActs : genetically encoded tools for perturbing the actin cytoskeleton in single cells

    NARCIS (Netherlands)

    Harterink, Martin; Santos Esteves da Silva, Marta; Will, Lena; Turan, Julia; Ibrahim, Adiljan; Lang, Alexander E; Van Battum, Eljo Y; Pasterkamp, R Jeroen; Kapitein, Lukas C; Kudryashov, Dmitri; Barres, Ben A; Hoogenraad, Casper C; Zuchero, J Bradley


    The actin cytoskeleton is essential for many fundamental biological processes, but tools for directly manipulating actin dynamics are limited to cell-permeable drugs that preclude single-cell perturbations. Here we describe DeActs, genetically encoded actin-modifying polypeptides, which effectively

  14. Identification of innate lymphoid cells in single-cell RNA-Seq data. (United States)

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


    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.

  15. Identification of soybean proteins from a single cell type: The root hair

    Energy Technology Data Exchange (ETDEWEB)

    Brechenmacher, Laurent; Nguyen, Tran H.; Hixson, Kim K.; Libault, Marc; Aldrich, Joshua T.; Pasa-Tolic, Ljiljana; Stacey, Gary


    Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.

  16. Recent advances in the development of single cell analysis-A review

    Czech Academy of Sciences Publication Activity Database

    Klepárník, Karel; Foret, František


    Roč. 800, OCT (2013), s. 12-21 ISSN 0003-2670 R&D Projects: GA MŠk(CZ) EE2.3.20.0182; GA ČR GAP206/11/2377 Institutional support: RVO:68081715 Keywords : single cell analysis * capillary electrophoresis * electrochemistry * microfluidic devices Subject RIV: CB - Analytical Chemistry , Separation Impact factor: 4.517, year: 2013

  17. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches (United States)

    Wu, Jincheng; Tzanakakis, Emmanuel S.


    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

  18. Economic Optimizing Control for Single-Cell Protein Production in a U-Loop Reactor

    DEFF Research Database (Denmark)

    Drejer, André; Ritschel, Tobias Kasper Skovborg; Jørgensen, Sten Bay


    The production of single-cell protein (SCP) in a U-loop reactor by a methanotroph is a cost efficient sustainable alternative to protein from fish meal obtained by over-fishing the oceans. SCP serves as animal feed. In this paper, we present a mathematical model that describes the dynamics of SCP...

  19. Noisy Response to Antibiotic Stress Predicts Subsequent Single-Cell Survival in an Acidic Environment. (United States)

    Mitosch, Karin; Rieckh, Georg; Bollenbach, Tobias


    Antibiotics elicit drastic changes in microbial gene expression, including the induction of stress response genes. While certain stress responses are known to "cross-protect" bacteria from other stressors, it is unclear whether cellular responses to antibiotics have a similar protective role. By measuring the genome-wide transcriptional response dynamics of Escherichia coli to four antibiotics, we found that trimethoprim induces a rapid acid stress response that protects bacteria from subsequent exposure to acid. Combining microfluidics with time-lapse imaging to monitor survival and acid stress response in single cells revealed that the noisy expression of the acid resistance operon gadBC correlates with single-cell survival. Cells with higher gadBC expression following trimethoprim maintain higher intracellular pH and survive the acid stress longer. The seemingly random single-cell survival under acid stress can therefore be predicted from gadBC expression and rationalized in terms of GadB/C molecular function. Overall, we provide a roadmap for identifying the molecular mechanisms of single-cell cross-protection between antibiotics and other stressors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Stability of Axially Compressed Single-Cell Mono-Symmetric Thin ...

    African Journals Online (AJOL)

    Compared with conventional structural columns, the pronounced role of instabilities complicates the behaviour and design of thin-walled columns. This study investigated the stability of axially compressed single-cell thin-walled column with mono-symmetric non-deformable cross-sections. The work involved a theoretical ...

  1. Single-cell screening of photosynthetic growth and lactate production by cyanobacteria

    NARCIS (Netherlands)

    Hammar, P.; Angermayr, S.A.; Sjostrom, S.L.; van der Meer, J.; Hellingwerf, K.J.; Hudson, E.P.; Joensson, H.N.


    BACKGROUND: Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. RESULTS: We present a method for high-throughput, single-cell

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

    Directory of Open Access Journals (Sweden)

    Anouchka eFievet


    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.

  3. Understanding gene expression variability in its biological context using theoretical and experimental analyses of single cells

    NARCIS (Netherlands)

    Kempe, H.


    Traditional gene expression studies have largely ignored cell-to-cell variability in transcription. Current methods allow for single cell analyses and have shown considerable variability in gene expression, even in populations of isogenic cells exposed to the same growth environment. In this thesis,

  4. Yeast single cell protein in the diet of Oreochromis niloticus (L ...

    African Journals Online (AJOL)


    Diets D10 to D50 had fish meal replaced systematically with yeast single cell protein (SCP) in the order 10, 20, 30, 40 and 50%, respectively. Trial feeding was ... Key word: microbial protein, Oreochromis niloticus, feeding, cost benefit, aquaculture. .... Supplementary Feeding for Production of Nile Tilapia, Silver Carp.

  5. Recent advances in the development of single cell analysis-A review

    Czech Academy of Sciences Publication Activity Database

    Klepárník, Karel; Foret, František


    Roč. 800, OCT (2013), s. 12-21 ISSN 0003-2670 R&D Projects: GA MŠk(CZ) EE2.3.20.0182; GA ČR GAP206/11/2377 Institutional support: RVO:68081715 Keywords : single cell analysis * capillary electrophoresis * electrochemistry * microfluidic devices Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.517, year: 2013

  6. Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells (United States)

    Zamani Esteki, Masoud; Dimitriadou, Eftychia; Mateiu, Ligia; Melotte, Cindy; Van der Aa, Niels; Kumar, Parveen; Das, Rakhi; Theunis, Koen; Cheng, Jiqiu; Legius, Eric; Moreau, Yves; Debrock, Sophie; D’Hooghe, Thomas; Verdyck, Pieter; De Rycke, Martine; Sermon, Karen; Vermeesch, Joris R.; Voet, Thierry


    Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones. PMID:25983246

  7. Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle. (United States)

    Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji


    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.

  8. Formation of monofluorocarbon compounds by single cell cultures of Glycine max growing on inorganic fluoride

    Energy Technology Data Exchange (ETDEWEB)

    Peters, R.A.; Shorthouse, M.


    Single cell cultures of Glycine max were exposed to sodium fluoride to determine the capacity for synthesis of fluoroacetate and fluorocitrate. This capacity had previously been observed in soybean plants and alfalfa. The results show that cell cultures of Glycine max can also synthesize these fluoroacids.

  9. Single cell cytometry of protein function in RNAi treated cells and in native populations

    Directory of Open Access Journals (Sweden)

    Hill Andrew


    Full Text Available Abstract Background High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models. Results We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level. Conclusion We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell

  10. Prognostic significance of tumor budding and single cell invasion in gastric adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Che K


    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

  11. 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.

  12. Comet and meteorite traditions of Aboriginal Australians (United States)

    Hamacher, Duane W.


    This research contributes to the disciplines of cultural astronomy (the academic study of how past and present cultures understand and utilise celestial objects and phenomena) and geomythology (the study of geological events and the formation of geological features described in oral traditions). Of the hundreds of distinct Aboriginal cultures of Australia, many have oral traditions rich in descriptions and explanations of comets, meteors, meteorites, airbursts, impact events, and impact craters. These views generally attribute these phenomena to spirits, death, and bad omens. There are also many traditions that describe the formation of meteorite craters as well as impact events that are not known to Western science.

  13. Workshop on Observations of Recent Comets (1990) (United States)

    Huebner, Walter F. (Editor); Wehinger, Peter A. (Editor); Rahe, Jurgen (Editor); Konno, Ichishiro (Editor)


    Potential interpretations are presented for observations of four comets: Brorsen-Metcalf (1989o), Okazaki-Levy-Rudenko (1989r), Aarseth-Brewington (1989a1), and Austin (1989o1). The relationship of minor species with each other and possible parents as well as with dust are being pursued in a number of investigations. Of particular interest are the abundance ratios of CH4 to CO and NH3 to N2. The need for closer collaboration betwen observing teams and modelers is examined. The need for dust size distribution as a function of cometocentric distance to be analyzed in closer collaboration between observers and modelers is discussed.

  14. The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

    Directory of Open Access Journals (Sweden)

    Bas-jan Van Der Leede


    Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

  15. Using measures of single-cell physiology and physiological state to understand organismic aging. (United States)

    Mendenhall, Alexander; Driscoll, Monica; Brent, Roger


    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.

  16. Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing. (United States)

    Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun


    Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

  17. Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment (United States)

    Asphahani, Fareid; Wang, Kui; Thein, Myo; Veiseh, Omid; Yung, Sandy; Xu, Jian; Zhang, Miqin


    The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell-based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that host individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell-based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.

  18. Mission to a comet that could save earth

    CERN Multimedia

    Utton, T


    Scientists are going to attempt to land a probe on the comet Wirtanen. The GBP640million unmanned craft will travel four billion miles before catching up with the comet Wirtanen and launching a robotic lander called Rosetta, on to its surface (1/2 page).

  19. Dormant Comets in the Near-Earth Asteroid Population

    NARCIS (Netherlands)

    Mommert, Michael; Harris, Alan W.; Mueller, Michael; Hora, Joseph L.; Trilling, David E.; Knight, Matthew; Bottke, William F.; Thomas, Cristina; Delbo', Marco; Emery, Josh P.; Fazio, Giovanni; Smith, Howard A.


    The population of near-Earth objects comprises active comets and asteroids, covering a wide range of dynamical parameters and physical properties. Dormant (or extinct) comets, masquerading as asteroids, have long been suspected of supplementing the near-Earth asteroid (NEA) population. We present a

  20. The Formation Age of Comets: Predicted Physical and Chemical Trends (United States)

    Nuth, J. A., III; Hill, H. G. M.


    The chemical composition of a comet has always been considered to be a function of where it formed in the nebula. We suggest that the most important factor in determining a comet's chemistry might actually be when it formed. Specific predictions are presented.

  1. Direct Detection of Extra-Solar Comets is Possible


    Jura, M.


    The dust tails of comets similar to Hale-Bopp can scatter as much optical light as does the Earth. Space-based observatories such as the Terrestrial Planet Finder or Darwin that will detect extra-solar terrestrial planets also will be able to detect extra-solar comets.

  2. Ensemble Properties of Comets in the Sloan Digital Sky Survey

    Energy Technology Data Exchange (ETDEWEB)

    Solontoi, Michael; /Adler Planetarium, Chicago; Ivezic, Zeljko; /Washington U., Seattle, Astron. Dept.; Juric, Mario; /Harvard Coll. Observ.; Becker, Andrew C.; /Washington U., Seattle, Astron. Dept.; Jones, Lynne; /Washington U., Seattle, Astron. Dept.; West, Andrew A.; /Boston U.; Kent, Steve; /Fermilab; Lupton, Robert H.; /Princeton U. Observ.; Claire, Mark; /Washington U., Seattle, Astron. Dept.; Knapp, Gillian R.; /Princeton U. Observ.; Quinn, Tom; /Washington U., Seattle, Astron. Dept. /Princeton U. Observ.


    We present the ensemble properties of 31 comets (27 resolved and 4 unresolved) observed by the Sloan Digital Sky Survey (SDSS). This sample of comets represents about 1 comet per 10 million SDSS photometric objects. Five-band (u, g, r, i, z) photometry is used to determine the comets colors, sizes, surface brightness profiles, and rates of dust production in terms of the Afp formalism. We find that the cumulative luminosity function for the Jupiter Family Comets in our sample is well fit by a power law of the form N(comets. The resolved comets show an extremely narrow distribution of colors (0.57 {+-} 0.05 in g - r for example), which are statistically indistinguishable from that of the Jupiter Trojans. Further, there is no evidence of correlation between color and physical, dynamical, or observational parameters for the observed comets.

  3. The exploration of Halley's comet - An example of international cooperation (United States)

    Rahe, Jurgen H.; Newburn, Ray L., Jr.


    The history of international cooperation in studies of comets started with observations in 1577 and 1680, when Tycho Brahe and Newton, respectively, collected position measurements made in different countries to determine the paths of the comets observed. In the fall of 1979, a worldwide Comet Halley watch was proposed. As a result of international cooperation, Comet Halley was explored during its recent appearance from the ground, earth orbit, Venus orbit, interplanetary space, and from within the comet itself. The various activities in space were coordinated by the ESA, the USSR Intercosmos, the Japanese ISAS, and NASA, through the Inter-Agency Consultative Group. The activities of the ground-based observers were coordinated by the International Halley Watch.

  4. Differential genotoxicity of ethyl methanesulphonate, N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš


    Roč. 538, - (2003), s. 171-179 ISSN 1383-5718 R&D Projects: GA ČR GA521/02/0400; GA MŠk LN00B030 Institutional research plan: CEZ:AV0Z5038910 Keywords : DNA damage * Nicotiana tabacum * Single cell gel electrophoresis (SCGE) Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.748, year: 2003


    Energy Technology Data Exchange (ETDEWEB)

    Feldman, Paul D., E-mail: [Department of Physics and Astronomy, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218 (United States)


    Observations of comets in the 905–1180 Å spectral band made with the Far Ultraviolet Spectroscopic Explorer in 2001 and 2004 show unusual features in the fluorescent emissions of CO and H{sub 2}. These include emission from a non-thermal high-J rotational population of CO and solar Lyα induced fluorescence from excited vibrational levels of H{sub 2}, both of which are attributed to the photodissociation of formaldehyde. In this paper we model the large number of observed H{sub 2} lines and demonstrate the dependence of the pumping on the heliocentric velocity of the comet and the solar line profiles. We also derive the rotational and vibrational populations of H{sub 2} and show that they are consistent with the results of laboratory studies of the photodissociation of H{sub 2}CO. In addition to the principal series of H i and O i, the residual spectrum is found to consist mainly of the Rydberg series of C i multiplets from which we derive the mean carbon column abundance in the coma. Fluorescent emissions from N i and N{sub 2} are also searched for.

  6. Active Polymer Gel Actuators

    Directory of Open Access Journals (Sweden)

    Shuji Hashimoto


    Full Text Available Many kinds of stimuli-responsive polymer and gels have been developed and applied to biomimetic actuators or artificial muscles. Electroactive polymers that change shape when stimulated electrically seem to be particularly promising. In all cases, however, the mechanical motion is driven by external stimuli, for example, reversing the direction of electric field. On the other hand, many living organisms can generate an autonomous motion without external driving stimuli like self-beating of heart muscles. Here we show a novel biomimetic gel actuator that can walk spontaneously with a wormlike motion without switching of external stimuli. The self-oscillating motion is produced by dissipating chemical energy of oscillating reaction. Although the gel is completely composed of synthetic polymer, it shows autonomous motion as if it were alive.

  7. Comet Hunters: A Citizen Science Project to Search for Comets in the Main Asteroid Belt (United States)

    Hsieh, Henry H.; Schwamb, Megan Elizabeth; Zhang, Zhi-Wei; Chen, Ying-Tung; Wang, Shiang-Yu; Lintott, Chris


    Fully automated detection of comets in wide-field surveys remains a challenge, as even highly successful comet-finding surveys like Pan-STARRS rely on a combination of both automated flagging algorithms and vetting by human eyes. To take advantage of the long-noted superiority of the human eye over computer algorithms in certain types of pattern recognition, particularly when dealing with a range of target morphologies of interest, we have created a citizen science website with the aim of allowing the general public to aid in the search for active asteroids, which are objects that occupy dynamically asteroidal orbits yet exhibit comet-like dust emission due to sublimation, impact disruption, rotational destabilization, or other effects. Located at, the Comet Hunters website was built using the Zooniverse Project Builder (, and displays images of known asteroids obtained either from archival data obtained between 1999 and 2014 by the Suprime-Cam wide-field imager mounted on the 8-m Subaru telescope on Mauna Kea in Hawaii, or more contemporary data obtained by the Hyper Suprime-Cam (HSC) wide-field imager also on the Subaru Telescope as part of the ongoing HSC Subaru Strategic Program (SSP) survey. By using observations from such a large-aperture telescope, most of which have never been searched for solar system objects, much less cometary ones, we expect that volunteers should be able to make genuinely scientifically significant discoveries, and also provide valuable insights into the potential and challenges of searching for comets in the LSST era. To date, over 13,000 registered volunteers have contributed 350,000 classifications. We will discuss the design and construction of the Comet Hunters website, and also discuss early results from the project.This work uses data generated via the platform, development of which was supported by a Global Impact Award from Google, and by the Alfred P. Sloan

  8. Perspectives on Comets, Comet-like Asteroids, and Their Predisposition to Provide an Environment That Is Friendly to Life. (United States)

    Bosiek, Katharina; Hausmann, Michael; Hildenbrand, Georg


    In recent years, studies have shown that there are many similarities between comets and asteroids. In some cases, it cannot even be determined to which of these groups an object belongs. This is especially true for objects found beyond the main asteroid belt. Because of the lack of comet fragments, more progress has been made concerning the chemical composition of asteroids. In particular, the SMASSII classification establishes a link between the reflecting spectra and chemical composition of asteroids and meteorites. To find clues for the chemical structure of comets, the parameters of all known asteroids of the SMASSII classification were compared to those of comet groups like the Encke-type comets, the Jupiter-family comets, and the Halley-type comets, as well as comet-like objects like the damocloids and the centaurs. Fifty-six SMASSII objects similar to comets were found and are categorized as comet-like asteroids in this work. Aside from the chemistry, it is assumed that the available energy on these celestial bodies plays an important role concerning habitability. For the determination of the available energy, the effective temperature was calculated. Additionally, the size of these objects was considered in order to evaluate the possibility of a liquid water core, which provides an environment that is more likely to support processes necessary to create the building blocks of life. Further study of such objects could be notable for the period of the Late Heavy Bombardment and could therefore provide important implications for our understanding of the inner workings of the prebiotic evolution within the Solar System since the beginning.

  9. Sample Preparation Methods Following CellSearch Approach Compatible of Single-Cell Whole-Genome Amplification: An Overview

    NARCIS (Netherlands)

    Swennenhuis, Joost Franciscus; Terstappen, Leonardus Wendelinus Mathias Marie; Kroneis, Thomas


    Single cells are increasingly used to determine the heterogeneity of therapy targets in the genome during the course of a disease. The first challenge using single cells is to isolate these cells from the surrounding cells, especially when the targeted cells are rare. A number of techniques have

  10. Live-Cell Imaging Tool Optimization To Study Gene Expression Levels and Dynamics in Single Cells of Bacillus cereus

    NARCIS (Netherlands)

    Eijlander, Robyn T.; Kuipers, Oscar P.

    Single-cell methods are a powerful application in microbial research to study the molecular mechanism underlying phenotypic heterogeneity and cell-to-cell variability. Here, we describe the optimization and application of single-cell time-lapse fluorescence microscopy for the food spoilage bacterium

  11. Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Dalton, Helen M.; Angels, Mark


    A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells: a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of Fluorescently labelled oligonucleotide probes to rRNA. The method...... allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells....

  12. 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

  13. 3D MR gel dosimetry with lung equivalent gel; 3D MR-Gel-Dosimetrie mit lungenaequivalentem Gel

    Energy Technology Data Exchange (ETDEWEB)

    Scherer, J.; Solleder, M.; Schiessl, I.; Bogner, L.; Herbst, M. [Klinik und Poliklinik fuer Strahlentherapie und Radioonkologie, Regensburg Univ. (Germany)


    The MR gel dosimetry is used to verify complex 3D treatment plans. Till now this method served only for dose evaluation in homogeneous phantoms. On the way to build a heterogeneous anthropomorphic gel phantom, a lung equivalent gel with the density 0.4 g/cm{sup 3} was developed. First experiments show a 1.55 times higher dose reponse in the low density gel (LD gel). The comparison of a dose distribution in a gel/LD gel/gel slab phantom with Monte Carlo calculations shows good agreement within 5%. More over the accuray of the measuring device magnetic resonance imager was studied in respect to the now exclusive digital image processing with the software MRD (MR dosimetry). Because of the dimensions of the Fricke gel phantom an artefact correction, based on the data from the unirradiated phantom proved to be essential. (orig.) [Deutsch] Die MR-Gel-Dosimetrie zur Verifikation komplexer 3D-Bestrahlungsplaene wurde bislang ausschliesslich in homogenen Phantomen durchgefuehrt. Auf dem Wege zum Bau eines inhomogenen Humanoid-Gel-Phantoms wurde ein lungenaequivalentes Gel mit der Dichte 0,4 g/cm{sup 3} entwickelt. Erste Messungen zeigen ein um den Faktor 1,55 hoeheres Ansprechvermoegen in dem low-density-Gel (LD-Gel). Der Vergleich einer gemessen Dosisverteilung in einem Gel/LD-Gel/Gel Schichtphantom als einfaches Thoraxmodell mit Monte-Carlo-Rechnungen zeigt eine gute Uebereinstimmung innerhalb 5%. Ausserdem wurden Untersuchungen zur Messgenauigkeit des Kernspintomographen im Rahmen der nun ausschliesslich digitalen Auswertung mit Hilfe des Programms MRD (MR-Dosimetrie) durchgefuehrt. Es zeigt sich, dass eine Artefaktkorrektur auf der Basis einer Messung des unbestrahlten Phantoms bei grossen Fricke-Gel-Phantomen notwendig ist. (orig.)

  14. A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs). (United States)

    Lanigan, Peter M P; Ninkovic, Tanja; Chan, Karen; de Mello, Andrew J; Willison, Keith R; Klug, David R; Templer, Richard H; Neil, Mark A A; Ces, Oscar


    We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

  15. Accurate analytic solution of chemical master equations for gene regulation networks in a single cell (United States)

    Huang, Guan-Rong; Saakian, David B.; Hu, Chin-Kun


    Studying gene regulation networks in a single cell is an important, interesting, and hot research topic of molecular biology. Such process can be described by chemical master equations (CMEs). We propose a Hamilton-Jacobi equation method with finite-size corrections to solve such CMEs accurately at the intermediate region of switching, where switching rate is comparable to fast protein production rate. We applied this approach to a model of self-regulating proteins [H. Ge et al., Phys. Rev. Lett. 114, 078101 (2015), 10.1103/PhysRevLett.114.078101] and found that as a parameter related to inducer concentration increases the probability of protein production changes from unimodal to bimodal, then to unimodal, consistent with phenotype switching observed in a single cell.

  16. Single cell Hi-C reveals cell-to-cell variability in chromosome structure (United States)

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


    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

  17. Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica (United States)

    Radecki, Guillaume; Nargeot, Romuald; Jelescu, Ileana Ozana; Le Bihan, Denis; Ciobanu, Luisa


    In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn2+ concentrations. We establish that Mn2+ accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution. PMID:24872449

  18. Study of molasses / vinasse waste ratio for single cell protein and total microorganisms

    Directory of Open Access Journals (Sweden)

    Marcia Luciana Cazetta


    Full Text Available Different molasses/ vinasse ratio were used as substrate to investigate single cell protein and total lipids production by five microorganisms: four yeasts strains: Candida lipolytica, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, a yeast isolated from vinasse lake (denominated LLV98 and a bacterium strain, Corynebacterium glutamicum. The media utilized were: a 50% molasses and 50% vinasse; b 25% molasses and 75% vinasse and c 75% molasses and 25% vinasse. The objective of this work was to study the growth of microorganisms and also evaluate protein and lipids content in the biomass obtained from these by-products. The highest single cell protein production was obtained by S. cerevisiae, 50.35%, followed by R. mucilaginosa, 41.96%. The lowest productions were obtained by C. glutamicum. The higher total lipids productions, more than 26%, were founded in molasses plus vinasse at 50%/50% by S. cerevisiae and C. glutamicum.

  19. Bioconductor workflow for single-cell RNA sequencing: Normalization, dimensionality reduction, clustering, and lineage inference. (United States)

    Perraudeau, Fanny; Risso, Davide; Street, Kelly; Purdom, Elizabeth; Dudoit, Sandrine


    Novel single-cell transcriptome sequencing assays allow researchers to measure gene expression levels at the resolution of single cells and offer the unprecendented opportunity to investigate at the molecular level fundamental biological questions, such as stem cell differentiation or the discovery and characterization of rare cell types. However, such assays raise challenging statistical and computational questions and require the development of novel methodology and software. Using stem cell differentiation in the mouse olfactory epithelium as a case study, this integrated workflow provides a step-by-step tutorial to the methodology and associated software for the following four main tasks: (1) dimensionality reduction accounting for zero inflation and over dispersion and adjusting for gene and cell-level covariates; (2) cell clustering using resampling-based sequential ensemble clustering; (3) inference of cell lineages and pseudotimes; and (4) differential expression analysis along lineages.

  20. Single-Cell Genomics: A Stepping Stone for Future Immunology Discoveries. (United States)

    Giladi, Amir; Amit, Ido


    The immunology field has invested great efforts and ingenuity to characterize the various immune cell types and elucidate their functions. However, accumulating evidence indicates that current technologies and classification schemes are limited in their ability to account for the functional heterogeneity of immune processes. Single-cell genomics hold the potential to revolutionize the way we characterize complex immune cell assemblies and study their spatial organization, dynamics, clonal distribution, pathways, function, and crosstalks. In this Perspective, we consider recent and forthcoming technological and analytical advances in single-cell genomics and the potential impact of those advances on the future of immunology research and immunotherapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Single-cell screening of photosynthetic growth and lactate production by cyanobacteria

    DEFF Research Database (Denmark)

    Hammar, Petter; Angermayr, S. Andreas; Sjostrom, Staffan L.


    Background: Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible.Results: We present a method for high-throughput, single......-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for L-lactate production, and sort strains with high productivity. We demonstrate the separation...... of low- and high-producing reference strains, as well as enrichment of a more productive L-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically...

  2. A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells (United States)

    Yang, Tie; Bragheri, Francesca; Nava, Giovanni; Chiodi, Ilaria; Mondello, Chiara; Osellame, Roberto; Berg-Sørensen, Kirstine; Cristiani, Ilaria; Minzioni, Paolo


    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 apparatus parameters before performing the cell-characterization experiments, including a non-destructive method to characterize the optical force distribution inside the microchannel. The chip was used to study important cell-mechanics parameters in two human breast cancer cell lines, MCF7 and MDA-MB231. Results indicate that MDA-MB231 has both higher acoustic compressibility and higher optical deformability than MCF7, but statistical analysis shows that optical deformability and acoustic compressibility are not correlated parameters. This result suggests the possibility to use them to analyze the response of different cellular structures. We also demonstrate that it is possible to perform both measurements on a single cell, and that the order of the two experiments does not affect the retrieved values.

  3. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

    KAUST Repository

    Perozziello, Gerardo


    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.

  4. Chasing the precursor of functional hematopoietic stem cells at the single cell levels in mouse embryos. (United States)

    Wang, Xiaochen; Gong, Yuemin; Ema, Hideo


    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.

  5. Lab-on-a-Chip Platforms for Biophysical Studies of Cancer with Single-Cell Resolution. (United States)

    Shukla, Vasudha C; Kuang, Tai-Rong; Senthilvelan, Abirami; Higuita-Castro, Natalia; Duarte-Sanmiguel, Silvia; Ghadiali, Samir N; Gallego-Perez, Daniel


    Recent cancer research has more strongly emphasized the biophysical aspects of tumor development, progression, and microenvironment. In addition to genetic modifications and mutations in cancer cells, it is now well accepted that the physical properties of cancer cells such as stiffness, electrical impedance, and refractive index vary with tumor progression and can identify a malignant phenotype. Moreover, cancer heterogeneity renders population-based characterization techniques inadequate, as individual cellular features are lost in the average. Hence, platforms for fast and accurate characterization of biophysical properties of cancer cells at the single-cell level are required. Here, we highlight some of the recent advances in the field of cancer biophysics and the development of lab-on-a-chip platforms for single-cell biophysical analyses of cancer cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Quantifying bacterial adhesion on antifouling polymer brushes via single-cell force spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Rodriguez-Emmenegger, Cesar; Janel, S.; de los Santos Pereira, Andres; Bruns, M.; Lafont, F.


    Roč. 6, č. 31 (2015), s. 5740-5751 ISSN 1759-9954 R&D Projects: GA ČR(CZ) GJ15-09368Y; GA MŠk(CZ) ED1.1.00/02.0109 Grant - others:OPPK(XE) CZ.2.16/3.1.00/21545 Program:OPPK Institutional support: RVO:61389013 Keywords : antifouling polymer brushes * single-cell force spectroscopy * bacterial adhesion Subject RIV: BO - Biophysics Impact factor: 5.687, year: 2015

  7. Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution. (United States)

    Sellix, Michael T; Currie, Jake; Menaker, Michael; Wijnen, Herman


    The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.

  8. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization


    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian


    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research oppor...

  9. Digital cell counting device integrated with a single-cell array.

    Directory of Open Access Journals (Sweden)

    Tatsuya Saeki

    Full Text Available In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2 in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2  = 0.99. This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

  10. Optimal Pilot and Payload Power Control in Single-Cell Massive MIMO Systems


    Cheng, Hei Victor; Björnson, Emil; Larsson, Erik G.


    This paper considers the jointly optimal pilot and data power allocation in single-cell uplink massive multiple-input-multiple- output systems. Using the spectral efficiency (SE) as performance metric and setting a total energy budget per coherence interval, the power control is formulated as optimization problems for two different objective functions: the weighted minimum SE among the users and the weighted sum SE. A closed form solution for the optimal length of the pilot sequence is derive...

  11. Single-cell sequencing reveals karyotype heterogeneity in murine and human malignancies. (United States)

    Bakker, Bjorn; Taudt, Aaron; Belderbos, Mirjam E; Porubsky, David; Spierings, Diana C J; de Jong, Tristan V; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Bradley, Allan; de Bont, Eveline S J M; van den Berg, Anke; Guryev, Victor; Lansdorp, Peter M; Colomé-Tatché, Maria; Foijer, Floris


    Chromosome instability leads to aneuploidy, a state in which cells have abnormal numbers of chromosomes, and is found in two out of three cancers. In a chromosomal instable p53 deficient mouse model with accelerated lymphomagenesis, we previously observed whole chromosome copy number changes affecting all lymphoma cells. This suggests that chromosome instability is somehow suppressed in the aneuploid lymphomas or that selection for frequently lost/gained chromosomes out-competes the CIN-imposed mis-segregation. To distinguish between these explanations and to examine karyotype dynamics in chromosome instable lymphoma, we use a newly developed single-cell whole genome sequencing (scWGS) platform that provides a complete and unbiased overview of copy number variations (CNV) in individual cells. To analyse these scWGS data, we develop AneuFinder, which allows annotation of copy number changes in a fully automated fashion and quantification of CNV heterogeneity between cells. Single-cell sequencing and AneuFinder analysis reveals high levels of copy number heterogeneity in chromosome instability-driven murine T-cell lymphoma samples, indicating ongoing chromosome instability. Application of this technology to human B cell leukaemias reveals different levels of karyotype heterogeneity in these cancers. Our data show that even though aneuploid tumours select for particular and recurring chromosome combinations, single-cell analysis using AneuFinder reveals copy number heterogeneity. This suggests ongoing chromosome instability that other platforms fail to detect. As chromosome instability might drive tumour evolution, karyotype analysis using single-cell sequencing technology could become an essential tool for cancer treatment stratification.

  12. T cell fate and clonality inference from single-cell transcriptomes. (United States)

    Stubbington, Michael J T; Lönnberg, Tapio; Proserpio, Valentina; Clare, Simon; Speak, Anneliese O; Dougan, Gordon; Teichmann, Sarah A


    We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.

  13. The workflow of single-cell expression profiling using quantitative real-time PCR

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Kubista, Mikael


    Roč. 14, č. 3 (2014), s. 323-331 ISSN 1473-7159 R&D Projects: GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : single-cell workflow * gene expression profiling * RT-qPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.516, year: 2014

  14. Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq (United States)


    individual cell types within human adipose tissue interact to regulate adipose tissue physiology . Specifically, we have developed the molecular and...AWARD NUMBER: W81XWH-15-1-0251 TITLE: “Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA...TYPE Annual 3. DATES COVERED 1 AUG 2016 - 31 Aug 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Evaluation of Human Adipose Tissue Stromal

  15. Heterogeneity of Astrocytes: From Development to Injury - Single Cell Gene Expression

    Czech Academy of Sciences Publication Activity Database

    Rusňáková, Vendula; Honsa, Pavel; Džamba, Dávid; Stahlberg, A.; Kubista, Mikael; Anděrová, Miroslava


    Roč. 8, č. 8 (2013), e69734 E-ISSN 1932-6203 R&D Projects: GA ČR GA13-02154S Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z50390703 Keywords : Single cell expression profiling * astrocytes * GenEx Subject RIV: EB - Genetics ; Molecular Biology; FH - Neurology (UEM-P) Impact factor: 3.534, year: 2013

  16. SCANPY: large-scale single-cell gene expression data analysis. (United States)

    Wolf, F Alexander; Angerer, Philipp; Theis, Fabian J


    SCANPY is a scalable toolkit for analyzing single-cell gene expression data. It includes methods for preprocessing, visualization, clustering, pseudotime and trajectory inference, differential expression testing, and simulation of gene regulatory networks. Its Python-based implementation efficiently deals with data sets of more than one million cells ( ). Along with SCANPY, we present ANNDATA, a generic class for handling annotated data matrices ( ).

  17. Comparative Studies of Polymer Electrolyte Membrane Fuel Cell Stacks and Single Cells (United States)


    in the Catalyst Layer and Effects of Both Perfluorosulfonate Ionomer and PTFE-Loaded Carbon on the Catalyst Layer of Polymer Electrolyte Fuel Cells support of this project. 12 References 1. T. F. Fuller, "Is a Fuel Cell in Your Future?" 77K Electrochemical Society Interface (Fall...ARMY RESEARCH LABORATORY mm^ n Comparative Studies of Polymer Electrolyte Membrane Fuel Cell Stacks and Single Cells Deryn Chu and Rongzhong

  18. Convolutional Deep Belief Networks for Single-Cell/Object Tracking in Computational Biology and Computer Vision


    Zhong, Bineng; Pan, Shengnan; Zhang, Hongbo; Wang, Tian; Du, Jixiang; Chen, Duansheng; Cao, Liujuan


    In this paper, we propose deep architecture to dynamically learn the most discriminative features from data for both single-cell and object tracking in computational biology and computer vision. Firstly, the discriminative features are automatically learned via a convolutional deep belief network (CDBN). Secondly, we design a simple yet effective method to transfer features learned from CDBNs on the source tasks for generic purpose to the object tracking tasks using only limited amount of tra...

  19. Single-cell gene-expression profiling and its potential diagnostic applications

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Kubista, Mikael; Aman, P.


    Roč. 11, č. 7 (2011), s. 735-740 ISSN 1473-7159 R&D Projects: GA ČR(CZ) GAP303/10/1338; GA ČR(CZ) GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : gene-expression profiling * RT-qPCR * single-cell gene-expression profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.859, year: 2011

  20. Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the Human Subgingival Crevice


    Ouverney, Cleber C.; Armitage, Gary C.; Relman, David A.


    Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells fr...